Systems for surface-enhanced affinity capture for desorption and detection of analytes

ABSTRACT

This invention is directed to systems containing probes for presenting an analyte to an energy source for desorption in methods of analytic detection, such as mass spectrometry. The probes have an immobilized affinity reagent which binds the analyte on their presenting surface.

BACKGROUND OF THE INVENTION

This invention relates generally to methods and apparatus for desorptionand ionization of analytes for the purpose of subsequent scientificanalysis by such methods, for example, as mass spectrometry (MS) orbiosensors. Generally, analysis by mass spectrometry involves thevaporization and ionization of a small sample of material, using a highenergy source, such as a laser, including a laser beam. The material isvaporized from the surface of a probe tip into the gas or vapor phase bythe laser beam, and, in the process, some of the individual moleculesare ionized by the gain of a proton. The positively charged ionizedmolecules are then accelerated through a short high voltage field andlet fly (drift) into a high vacuum chamber, at the far end of which theystrike a sensitive detector surface. Since the time-of-flight is afunction of the mass of the ionized molecule, the elapsed time betweenionization and impact can be used to determine the molecule's masswhich, in turn, can be used to identify the presence or absence of knownmolecules of specific mass.

All known prior art procedures which present proteins or other largebiomolecules on a probe tip for laser desorption/ionizationtime-of-flight mass spectrometry (TOF) rely on the preparation of acrystalline solid mixture of the protein or other analyte molecule in alarge molar excess of acidic matrix material deposited on the baresurface of a metallic probe tip. (The sample probe tip typically ismetallic, either stainless steel, nickel plated material or platinum).Embedding the analyte in such a matrix was thought to be necessary inorder to prevent the destruction of analyte molecules by the laser beam.The laser beam strikes the solid mixture on the probe tip and its energyis used to vaporize a small portion of the matrix material along withsome of the embedded analyte molecules. Without the matrix, the analytemolecules are easily fragmented by the laser energy, so that the mass,and identity, of the original macromolecule is very difficult orimpossible to determine.

This prior art procedure has several limitations which have preventedits adaptation to automated protein or other macrobiological molecularanalysis. First, in a very crude sample it is necessary to partiallyfractionate (or otherwise purify the sample as much as possible) toeliminate the presence of excessive extraneous materials in thematrix/analyte crystalline or solid mixture. The presence of largequantities of components may depress the ion signal (either desorption,ionization and/or detection) of the targeted analyte. Such purificationis time-consuming, expensive, typically results in low recovery (orcomplete loss) of the analyte, and would be very difficult to do in anautomated analyzer.

Second, while the amount of analyte material needed for analysis by theprior art method is not large (typically in a picomole range), in somecircumstances, such as tests on pediatric patients, analyte fluids areavailable only in extremely small volumes (microliters) and may beneeded for performing several different analyses. Therefore, even thesmall amount (i.e., volume) needed for preparation of the analyte/matrixcrystalline mixture for a single analysis may be significant. Also, onlya tiny fraction (a few thousandths or less) of analyte used in preparingthe solid analyte/matrix mixture for use on the probe tip is actuallyconsumed in the desorption or mass spectrometric analysis. Anyimprovement in the prior art procedure which would make it possibleto 1) use much less analyte, 2) to locate the analyte or multipleanalytes on the probe tip or surface in a predetermined location, 3) toperform repeated analyses of the same aliquot of analyte (e.g., beforeand after one or more chemical and or enzymatic reactions), and 4) toconduct the test in a more quantitative manner, would be highlyadvantageous in many clinical areas.

Third, the analyte protein, or other macromolecule, used in preparingthe solid solution of analyte/matrix for use on the probe tip is notsuitable for any subsequent chemical tests or procedures because it isbound up (i.e., embedded) in the matrix material. Also, all of thematrix material used to date is strongly acidic, so that it wouldadversely affect many chemical reactions which might be attempted on themixture in order to modify the analyte molecules for subsequentexamination. Any improvement in the procedure which made it possible toconduct subsequent chemical modifications or reactions on the analytemolecules, without removing them from the matrix or the probe tip orwithout "matrix" altogether, would be of enormous benefit to researchersand clinicians.

The first successful molecular mass measurements of intact peptides andsmall proteins (only up to about 15 kDa) by any form of massspectrometry were made by bombarding surfaces with high energy particles(plasma desorption and fast atom bombardment mass spectrometry); thisbreakthrough came in 1981 and 1982. Improvements came in 1985 and 1986,however, yield (signal intensities), sensitivity, precision, and massaccuracy remained relatively low. Higher molecular mass proteins (about20 to 25 kDa) were not observed except on rare occasions; proteinsrepresenting average molecular weights (approximately 70 kDa) were notever observed with these methods. Thus, evaluation of most proteins bymass spectrometry remains unrealized.

In 1988, Hillenkamp and his coworkers used UV laser desorptiontime-of-flight mass spectrometry and discovered that when proteins ofrelatively high molecular mass were deposited on the probe tip in thepresence of a very large molar excess of an acidic, UV absorbingchemical matrix (nicotinic acid) they could be desorbed in the intactstate. This new technique is called matrix-assisted laserdesorption/ionization (MALDI) time-of-flight mass spectrometry. Notethat laser desorption time-of-flight mass spectrometry (without thechemical matrix) had been around for some time, however, there waslittle or no success determining the molecular weights of large intactbiopolymers such as proteins and nucleic acids because they werefragmented (destroyed) upon desorption. Thus, prior to the introductionof a chemical matrix, laser desorption mass spectrometry was essentiallyuseless for the detection of specific changes in the mass of intactmacromolecules. Note that the random formation of matrix crystals andthe random inclusion of analyte molecules in the solid solution is priorart.

There are a number of problems and limitations with the prior artmethods. For example, previously, it has been found that it is difficultto wash away contaminants present in analyte or matrix. Other problemsinclude formation of analyte-salt ion adducts, less than optimumsolubility of analyte in matrix, unknown location and concentration ofanalyte molecules within the solid matrix, signal (molecular ion)suppression "poisoning" due to simultaneous presence of multiplecomponents, and selective analyte desorption/ionization. Priorinvestigators, including Karas and Hillenkamp have reported a variety oftechniques for analyte detection using mass spectroscopy, but thesetechniques suffered because of inherent limitations in sensitivity andselectivity of the techniques, specifically including limitations indetection of analytes in low volume, undifferentiated samples.(Hillenkamp, Bordeaux Mass Spectrometry Conference Report, pp. 354-62(1988); Karas and Hillenkamp, Bordeaux Mass Spectrometry ConferenceReport, pp. 416-17 (1988); Karas and Hillenkamp, Analytical Chemistry,60:2299-2301 (1988); Karas, et al., Biomed. Environ. Mass Spectrum18:841-843 (1989). The use of laser beams in time-of-flight massspectrometers is shown, for example, in U.S. Pat. Nos. 4,694,167;4,686,366, 4,295,046, and 5,045,694, incorporated by reference.

The successful volatilization of high molecular weight biopolymers,without fragmentation, has enabled a wide variety of biologicalmacromolecules to be analyzed by mass spectrometry. More importantlyperhaps, it has illustrated the potential of using mass spectrometrymore creatively to solve problems routinely encountered in biologicalresearch. Most recent attention has been focused on the utility ofmatrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)mass spectrometry (MS), largely because it is rapid (min), sensitive (<pmol sample required), and permits complex mixtures to be analyzed.

Although MALDI-TOF MS continues to be useful for the staticdetermination/verification of mass for individual analytes, in the caseof biopolymers, it is often differences in mass that provide the mostimportant information about unknown structures. Thus, for routine use instructural biology, an unfortunate limitation of the MALDI-TOF MStechnique relates to sample preparation and presentation (deposition) onan inert probe element surface, specifically, the requirement thatanalytes be embedded (i.e., co-solidified) on the probe surface in afreshly prepared matrix of crystalline organic acid. The randomdistribution of analyte in a heterogeneous display of crystal matrix onthe probe element surface requires the deposition of far more analyte orsample than is needed for the laser desorption process, even for thecollection of more than adequate mass spectra (e.g., multiple sets of100 shots each). The remaining portion of the analyte is usually notrecovered for additional analyses or subsequent characterizations. Eventhough 1 to 10 pmol (sometimes less) of analyte are typically requiredfor deposition on the probe surface, it has been estimated that lessthan a few attomoles are consumed during laser desorption. Thus, only 1part in 10⁵ or 10⁶ of the applied analyte may be necessary; the rest islost.

Another important loss of potential data associated with the embeddingof analyte in a solid matrix is the reduction or the completeelimination of ability to perform subsequent chemical and/or enzymaticmodifications to the embedded analyte (e.g., protein or DNA) remainingon the probe surface. Only another aliquot of analyte, or the ability torecover the embedded analyte free of matrix (difficult with lowrecovery), allows what we now refer to as differential mass spectrometryto be performed to derive structural data.

In addition, there has been limited application of MS in biologicalfields, likely due to the fact that many biologists and clinicians areintimidated by MS and/or skeptical in regard to its usefulness. Further,MS is perceived as inaccessible or too costly, particularly because SDSpolyacrylamide gel electrophoresis is an adequate substitute in someinstances where MALDI would be applied (e.g., separation of crudebiological fluids). In addition, MALDI has had little exposure inbiological and clinical journals.

SUMMARY OF THE INVENTION

An object of the invention is to provide improved methods, materialscomposition and apparatus for coupled adsorption, desorption andionization of multiple or selected analytes into the gas (vapor) phase.

Another object is to provide a method and apparatus foraffinity-directed detection of analytes, including desorption andionization of analytes in which the analyte is not dispersed in a matrixsolution or crystalline structure but is presented within, on or abovean attached surface of energy absorbing "matrix" material throughmolecular recognition events, in a position where it is accessible andamenable to a wide variety of chemical, physical and biologicalmodification or recognition reactions.

Another object is to provide such a method and apparatus in which theanalyte material is chemically bound or physically adhered to asubstrate forming a probe tip sample presenting surface.

A further object is to provide means for the modification of samplepresenting surfaces with energy-absorbing molecules to enable thesuccessful desorption of analyte molecules without the addition ofexogenous matrix molecules as in prior art.

A further object is to provide the appropriate density ofenergy-absorbing molecules bonded (covalently or noncovalently) in avariety of geometries such that mono layers and multiple layers ofattached energy-absorbing molecules are used to facilitate thedesorption of analyte molecules of varying masses.

A further object is to provide multiple combinations of surfacesmodified with energy-absorbing molecules, affinity-directed analytecapture devices, phototubes, etc.

An additional object is to provide such a method and apparatus in whichthe substrate forming the probe tip or other sample presenting surfaceis derivatized with one or more affinity reagents (a variety ofdensities and degrees of amplification) for selective bonding withpredetermined analytes or classes of analytes.

A further object is to provide such a system in which the affinityreagent chemically bonds or biologically adheres to the target analyteor class of analytes.

A still further object is to provide a method and apparatus fordesorption and ionization of analytes in which unused portion of theanalytes contained on the presenting surface remain chemicallyaccessible, so that a series of chemical, enzymatic or physicaltreatments of the analyte may be conducted, followed by sequentialanalyses of the modified analyte.

A further object is to provide a method and apparatus for the combinedchemical or enzymatic modifications of target analytes for the purposeof elucidating primary, secondary, tertiary, or quaternary structure ofthe analyte and its components.

Another object is to provide a method and apparatus for desorption andionization of analyte materials in which cations other than protons (H⁺)are utilized for ionization of analyte macromolecules.

Thus, in accomplishing the foregoing objects, there is provided inaccordance with the present invention, an apparatus for measuring themass of an analyte molecule of an analyte sample by means of massspectrometry, said apparatus comprising a spectrometer tube; a vacuummeans for applying a vacuum to the interior of said tube; electricalpotential means within the tube for applying an accelerating electricalpotential to desorbed analyte molecules from said analyte sample; samplepresenting means removably insertable into said spectrometer tube, forpresenting said analyte sample in association with surface associatedmolecule for promoting desorption and ionization of said analytemolecules, wherein said surface molecule is selected from the groupconsisting of energy absorbing molecule, affinity capture device,photolabile attachment molecule and combination thereof; an analytesample deposited on said sample presenting means in association withsaid surface associated molecules, whereby at least a portion of saidanalyte molecules not consumed in said mass spectrometry analysis willremain accessible for subsequent chemical, biological or physicalanalytical procedures; laser beam means for producing a laser beamdirected to said analyte sample for imparting sufficient energy todesorb and ionize a portion of said analyte molecules from said analytesample; and detector means associated with said spectrometer tube fordetecting the impact of accelerated ionized analyte molecules thereon.

In addition, in accomplishing the foregoing objects, there is providedin accordance with the present invention, a method in mass spectrometryto measure the mass of an analyte molecule, said method comprising thesteps of: derivitizing a sample presenting surface on a probe tip facewith an affinity capture device having means for binding with an analytemolecule; exposing said derivitized probe tip face to a source of saidanalyte molecule so as to bind said analyte molecule thereto; placingthe derivitized probe tip with said analyte molecules bound thereto intoone end of a time-of-flight mass spectrometer and applying a vacuum andan electric field to form an accelerating potential within thespectrometer; striking at least a portion of the analyte molecules boundto said derivitized probe tip face within the spectrometer with one ormore laser pulses in order to desorb ions of said analyte molecules fromsaid tip; detecting the mass of the ions by their time of flight withinsaid mass spectrometer; and displaying such detected mass.

Further, in accomplishing the foregoing objects, there is provided inaccordance with the present invention, a method of measuring the mass ofanalyte molecules by means of laser desorption/ionization,time-of-flight mass spectrometry in which an energy absorbing materialis used in conjunction with said analyte molecules for facilitatingdesorption and ionization of the analyte molecules, wherein theimprovement comprises presenting the analyte molecules on or above thesurface of the energy absorbing material, wherein at least a portion ofthe analyte molecules not desorbed in said mass spectrometry analysisremain chemically accessible for subsequent analytical procedures.

Additionally, in accomplishing the foregoing objects, there is providedin accordance with the present invention, an apparatus for facilitatingdesorption and ionization of analyte molecules, said apparatuscomprising: a sample presenting surface; and surface associatedmolecules, wherein said surface associated molecules are selected fromthe group consisting of energy absorbing molecule, affinity capturedevice, photolabile attachment molecule and combination thereof, saidsurface associated molecules associated with said sample presentingsurface and having means for binding with said analyte molecules.

Further, there is provided a method for capturing analyte molecules on asample presenting surface and desorbing/ionizing said captured analytemolecules from said sample presenting surface for subsequent analysis,said method comprising: derivitizing said sample presenting surface withan affinity capture device or photolabile attachment molecule havingmeans for binding with said analyte molecules; exposing said derivitizedsample present surface to a sample containing said analyte molecules;capturing said analyte molecules on said derivitized sample presentingsurface by means of said affinity capture device or photolabileattachment molecule; and exposing said analyte molecules, while bound tosaid derivitized sample presenting surface by means of said affinitycapture device or photolabile attachment molecule, to an energy or lightsource to desorb at least a portion of said analyte molecules from saidsurface.

Additionally, in accordance with the present invention, there isprovided a method for preparing a surface for presenting analytemolecules for analysis, said method comprising: providing a substrate onsaid surface for supporting said analyte; derivitizing said substratewith an affinity capture device or photolabile attachment moleculehaving means for selectively bonding with said analyte; and a means fordetecting said analyte molecules bonded with said affinity capturedevice or photolabile attachment molecule.

Further, in accomplishing the foregoing objects, there is provided inaccordance with the present invention, a sample probe for promotingdesorption of intact analytes into the gas phase comprising: a samplepresenting surface; and an energy absorbing molecule associated withsaid sample presenting surface, wherein said sample probe promotesdesorption of an intact analyte molecule positioned on, above or betweenthe energy absorbing molecules when said sample probe is impinged by anenergy source. Further, the energy absorbing molecule in the probe isselected from the group consisting of cinnamamide, cinnamyl bromide,2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid.

Additionally, in accomplishing the foregoing objects, there is providedin accordance with the present invention, a sample probe for desorptionof intact analyte into the gas phase, comprising: a sample presentationsurface; and a surface associated molecule wherein said surfaceassociated molecule is a photolabile attachment molecule having at leasttwo binding sites, wherein at least one site is bound to the samplepresentation surface and at least one site is available to bind ananalyte and wherein the analyte binding site is photolabile.

In addition, in accomplishing the foregoing objects there is provided inaccordance with the present invention, a sample probe for promotingdesorption of intact analytes into the gas phase comprising: a samplepresentation surface; and either

a mixture of at least two different molecules selected from the groupconsisting of an affinity capture device, an energy absorbing moleculeand a photolabile attachment molecule associated with said samplepresentation surface; wherein when an analyte is associated with saidsample probe, said sample probe promotes the transition of the analyteinto the gas phase when said sample probe is impinged by an energysource; or at least two different affinity capture devices associatedwith said sample presentation surface; wherein, when said sample probeis impinged by an energy source, said sample probe promotes thetransition of an analyte molecule into the gas phase at different ratesdepending on the affinity capture device associated with said analytemolecule.

In addition, in accomplishing the foregoing objects there is provided inaccordance with the present invention, a sample probe for promotingdesorption of intact analyte into the gas phase, comprising: a samplepresentation surface; and either a surface associated molecule, whereinsaid surface associated molecule can function both as an energyabsorbing molecule and as an affinity capture device; or a surfaceassociated molecule wherein said surface associated molecule is aphotolabile attachment molecule having at least two binding sites,wherein at least one site is bound to the sample presentation surfaceand at least one site is available to bind an analyte and wherein theanalyte binding site is photolabile.

Additionally, there is provided in the present invention, a method inmass spectrometry to measure the mass of an analyte molecule, saidmethod comprising the steps of: derivitizing a sample presenting surfaceon a probe tip face with a photolabile attachment molecule (PAM),wherein said PAM has at least two binding sites, one binding site bindsto the sample presenting surface and at least one binding site isavailable for binding with an analyte molecule; exposing saidderivitized probe tip face to a source of said analyte molecule so as tobind said analyte molecule thereto; placing the derivitized probe tipwith said analyte molecules bound thereto into one end of atime-of-flight mass spectrometer and applying a vacuum and an electricfield to form an accelerating potential within the spectrometer;striking at least a portion of the analyte molecules bound to saidderivitized probe tip face within the spectrometer with one or morelaser pulses in order to desorb ions of said analyte molecules from saidtip; detecting the mass of the ions by their time of flight within saidmass spectrometer; and displaying such detected mass.

In addition, there is provided a method of measuring the mass of analytemolecules by means of laser desorption/ionization, time-of-flight massspectrometry in which a photolabile attachment molecule (PAM) is used inconjunction with said analyte molecules for facilitating desorption andionization of the analyte molecules, the improvement comprising:presenting the analyte molecules on or above the surface of the PAM,wherein at least a portion of the analyte molecules not desorbed in saidmass spectrometry analysis remain chemically accessible for subsequentanalytical procedures.

There is further provided in accordance with the present invention, asample probe for promoting of differential desorption of intact analyteinto the gas phase, comprising: a sample presentation surface; and atleast two different photolabile attachment molecules associated withsaid sample presentation surface; wherein, when said sample probe isimpinged by an energy source, said sample probe promotes the transitionof an analyte molecule into the gas phase at different rates dependingon the photolabile attachment molecule associated with said analytemolecule.

Additionally, there is provided in accordance with the presentinvention, a sample probe for promoting desorption of intact analytesinto the gas phase comprising: a sample presenting surface; and aphotolabile attachment molecule associated with said sample presentingsurface; wherein, when said sample probe is impinged by an energysource, said sample probe promotes the transition of an intact analytemolecule into the gas phase.

Further, there is provided in accordance with the present invention, amethod for biopolymer sequence determination comprising the steps of:binding a biopolymer analyte to probe tip containing a sample presentingsurface having a surface selected molecule selected from the groupconsisting of an energy absorbing molecule, an affinity capture device,a photolabile attachment molecule and a combination thereof; desorptionof biopolymer analyte in mass spectrometry analysis, wherein at least aportion of said biopolymer is not desorbed from the probe tip; analyzingthe results of the desorption modifying the biopolymer analyte stillbound to the probe tip; and repeating the desorption, analyzing andmodifying steps until the biopolymer is sequenced.

Other and further objects, features and advantages will be apparent andthe invention more readily understood from a reading of the followingspecification and by reference to the accompanying drawings forming apart thereof, wherein the examples of the presently preferredembodiments of the invention are given for the purposes of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects and advantages of the invention will beapparent from the following specification and from the accompanyingdrawings.

FIG. 1 (upper profile) shows the mass spectrum of the three peptides(human histidine rich glycoprotein metal-binding domains (GHHPH)₂ G(1206 Da), (GHHPH)₅ G (2904 Da), and human estrogen receptordimerization domain (D473-L525) (6168.4 Da)) desorbed in the presence ofneutralized energy absorbing molecules (sinapinic acid, pH 6.2). FIG. 1(lower profile) shows the sequential in situ metal (Cu)-binding of thepeptides in the presence of neutral energy absorbing molecules.

FIG. 2 (top profile) shows the mass spectrum of the human caseinphosphopeptide (5P, 2488 Da) desorbed in the presence of neutralizedenergy absorbing molecules (sinapinic acid, pH 6.5). FIG. 2 (second fromtop profile) shows the sequential in situ 5 min alkaline phosphatasedigestion to remove phosphate groups from the phosphopeptide. FIG. 2(third from top profile) shows the mass spectrum of the phosphopeptideafter further in phosphatase digestion in the presence of acidic energyabsorbing molecules (2,5 dihydroxybenzoic acid, pH 2) as described inprior art.

FIG. 3 shows a composite mass spectra of the (GHHPH)₅ G peptide (2904Da) before (lower profile) and after (upper profile) in situ digestionby carboxypeptidase P in the presence of neutralized energy absorbingmolecules (sinapinic acid, pH 6.2).

FIG. 4 shows a composite matrix-assisted laser desorption mass spectraof peptide mixtures desorbed from solid glass, polypropylene-coatedstainless steel, polystyrene-coated stainless steel and solid nylonprobe elements.

SEAC

FIG. 5A, top profile, shows the mass spectrum of sperm activating factor(933 Da) and neurotensin (1655 Da) (and their multiple Na-adducts) inthe peptide solution unadsorbed by the IDA-Cu(II) surface. FIG. 5A,middle profile, shows the mass spectrum of angiotensin I (1296.5 Da)plus Na-adduct peaks that were selectively adsorbed on the IDA-Cu(II)surface. FIG. 5A, bottom profile, and FIG. 5B, bottom profile, show themass spectrum of the same angiotensin I adsorbed on IDA-Cu(II) afterwater wash. FIG. 5B, middle profile, shows the sequential in situcopper-binding (1 and 2 Cu) by affinity adsorbed angiotensin I. FIG. 5B,top profile, shows the sequential in situ trypsin digestion of theaffinity adsorbed angiotensin I.

FIG. 6 shows the mass spectrum of myoglobin (4 to 8 fmole) affinityadsorbed on IDA-Cu(II) surface.

FIG. 7 (top profile) shows the mass spectrum of synthetic casein peptide(1934 Da) with multiple phosphorylated forms affinity adsorbed from acrude mixture on TED-Fe(III) surface. After sequential in situ alkalinephosphatase digestion, only the original nonphosphorylated form remained(lower profile).

FIG. 8, bottom profile, shows the mass spectrum of total proteins ininfant formula. FIG. 8, second from bottom profile, shows the massspectrum of phosphopeptides in infant formula affinity adsorbed onTED-Fe(III) surface. FIG. 8, second from top profile, shows the massspectrum of total proteins in gastric aspirate of preterm infantobtained after feeding the infant formula. FIG. 8, top profile, showsthe mass spectrum of phosphopeptides in the gastric aspirate affinityadsorbed on TED-Fe(III) surface.

FIG. 9A shows the composite mass spectra of human and bovinehistidine-rich glycoprotein adsorbed on IDA-Cu(II) surface before andafter N-glycanase digestion. The mass shifts represent the removal ofcarbohydrate from the respective glycoproteins. FIG. 9B shows thecomposite mass spectra of trypsin digested peptides from thedeglycosylated proteins of the two species (top profile for humanprotein, second from bottom profile for bovine protein) and in situCu(II)-binding of the trypsin digested peptides of the two species(second from top profile for human protein, bottom profile for bovineprotein; the numbers 1, 2 indicate the number of copper bound). FIG. 9Cshows that one such Cu(II)-binding peptide (bottom profile) has at least4 His residues which are specifically modified by diethylpyrocarbonateto form 4 N-carbethoxy-histidyl adducts (1-4, top profile). FIG. 9Dshows the partial C-terminal sequence of the major Cu-binding peptide inthe bovine histidine rich glycoprotein.

FIG. 10 (bottom profile) shows the mass spectrum of rabbit anti-humanlactoferrin immunoglobulin alone (control) affinity adsorbed on sheepanti-rabbit IgG paramagnetic surface. The top profile shows the massspectrum of human lactoferrin and rabbit anti-human lactoferrinimmunoglobulin complex affinity adsorbed on sheep anti-rabbit IgGparamagnetic surface.

A FIG. 11A shows the mass spectrum of human lactoferrin affinityadsorbed from preterm infant urine on a anti-human lactoferrinimmunoglobulin nylon surface.

FIG. 11B shows the equivalent mass spectrum of whole preterm infanturine containing 1 nmole/ml of lactoferrin.

FIG. 12 (lower profile) shows the mass spectrum of pure bovine histidinerich glycoprotein. The upper profile shows the mass spectrum of bovinehistidine rich glycoprotein and fragments affinity adsorbed from bovinecolostrum on anti-bovine histidine rich glycoprotein immunoglobulinsurface.

FIG. 13 shows the composite mass spectra of the peptides of folliclestimulating hormone recognized by the different anti-folliclestimulating hormone antibodies.

FIG. 14 shows the mass spectrum of human lactoferrin affinity adsorbedon a single bead of single-stranded DNA agarose deposited on a 0.5 mmdiameter probe element.

FIG. 15 shows the mass spectrum of human lactoferrin affinity adsorbedfrom preterm infant urine on single-stranded DNA surface

FIG. 16A shows the composite mass spectra of the total proteins in humanduodenal aspirate (lower profile) and the trypsin affinity adsorbed fromthe aspirate on a soybean trypsin inhibitor surface (upper profile).FIG. 16B stows the mass spectrum of trypsin affinity adsorbed from 1 ulof aspirate on a soybean trypsin inhibitor nylon surface.

FIG. 17A shows the mass spectrum of biotinylated insulin affinityadsorbed from human urine on a Streptavidin surface. FIG. 17B shows themass spectrum of biotinylated insulin affinity adsorbed from humanplasma on a Streptavidin surface.

FIG. 18 (upper profile) shows the mass spectrum of total proteins inhuman serum. FIG. 18 (lower profile) shows the mass spectrum of serumalbumin affinity adsorbed from human serum on a Cibacron-blue surface.

SEND

FIG. 19 shows the molecular structure of surface bound cinnamamide; Rrepresents the surface plus cross-linker.

FIG. 20 (upper profile) shows the mass spectrum of peptide mixturesdesorbed from surface bound cinnamamide. FIG. 20 (lower profile) showsthe mass spectrum of the same peptide mixtures with free cinnamamide.

FIG. 21 shows the molecular structure of surface bound cinnamyl bromide;R represents the surface plus cross-linker.

FIG. 22 (upper profile) shows the mass spectrum of peptide mixturesdesorbed from surface bound cinnamyl bromide. FIG. 22 (lower profile)shows the mass spectrum of the same peptide mixtures with free cinnamylbromide.

FIG. 23 shows the molecular structure of surface boundMAP-dihydroxybenzoic acid; R represents the surface plus cross-linker.

FIG. 24 (upper profile) shows the mass spectrum of peptide mixturesdesorbed from surface bound MAP alone. FIG. 24 (lower profile) shows themass spectrum of the same peptide mixtures desorbed from surface boundMAP-dihydroxybenzoic acid.

FIG. 25A shows the mass spectrum (1,200-50,000 m/z region) of myoglobindesorbed from surface bound α-cyano-4-hydroxycinnamic acid. FIG. 25Bshows the same mass spectrum in the low mass region (0-1200 m/z).

FIG. 26 shows the molecular structure of energy absorbing moleculesbound to polyacrylamide or nylon or acrylic surface via glutaraldehydeactivation.

FIG. 27 shows the molecular structure of energy absorbing moleculesbound to polyacrylamide or nylon or acrylic surface via divinyl sulfoneactivation.

FIG. 28 shows the molecular structure of energy absorbing moleculesbound to polyacrylamide or nylon or acrylic surface viadicyclohexylcarbodiimide activation.

FIG. 29 shows the molecular structure of energy absorbing moleculesbound to polyacrylamide or nylon or acrylic surface with multipleantigenic peptide via dicyclohexylcarbodiimide activation.

FIG. 30 shows the molecular structure of thiosalicylic acid bound toiminodiacetate (IDA)-Cu(II) surface.

FIG. 31 shows the mass spectrum of human estrogen receptor dimerizationdomain desorbed from thiosalicylic acid-IDA-Cu(II) surface.

FIG. 32 shows the molecular structure of α-cyano-4-hydroxycinnamic acidbound to DEAE surface.

FIG. 33A shows the mass spectrum of human estrogen receptor dimerizationdomain desorbed from sinapinic acid-DEAE surface. FIG. 33B shows themass spectrum of myoglobin desorbed from α-cyano-4-hydroxycinnamic acidDEAE surface.

FIG. 34 shows the molecular structure of α-cyano-4hydroxycinnamic acidbound to polystyrene surface.

SEPAR

FIG. 35 shows the C-terminal sequence analysis of surface immobilizedvia photolytic bond histidine rich glycoprotein metal binding domain.

DETAILED DESCRIPTION OF THE INVENTION

It will be apparent to one skilled in the art that various substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and the spirit of the invention.

The development of new MS probe element compositions with surfaces thatallow the probe element to actively participate in the capture anddocking of specific analytes has recently defined several newopportunities in the area now being described as Affinity MassSpectrometry (AMS). In brief, several types of new MS probe elementshave been designed (Hutchens and Yip, Rapid Commun Mass Spectrom, 7:576-580 (1993)) with Surfaces Enhanced for Affinity Capture (SEAC). Todate, SEAC probe elements have been used successfully to retrieve andtether different classes of biopolymers, particularly proteins, byexploiting what is known about protein surface structures andbiospecific molecular recognition.

Progress in structural biology continues to be limited by the inabilityto obtain biopolymer sequence information at an acceptable rate or levelof sensitivity. By utilizing the methods and apparatus of the presentinvention, it has been demonstrated that AMS provides an opportunity torelieve this limitation. Because the immobilized affinity capturedevices on the MS probe element surface (i.e., SEAC) determines thelocation and affinity (specificity) of the analyte for the probesurface, the subsequent analytical AMS process is much more efficientfor several reasons. First, the location of analyte on the probe elementsurface is predetermined. Thus, the subsequent desorption is no longerdependent on a random search of the probe surface matrix field with theincident laser beam. Second, analyte detection sensitivity (and dynamicrange) is increased because molecular ionization suppression effectsoften observed with complex mixtures are eliminated. Third, the tetheredanalyte that is not actually consumed by the initial laser-induceddesorption process remains available for subsequent analyses. Ifexogenous matrix was used to promote analyte desorption, it is removed,in most cases, without loss of the tethered analyte. The remaininganalyte can then be chemically and/or enzymatically modified directly insitu (i.e., while still on the probe element). When analyzed again by MSto determine differences in mass, specific structural details arerevealed. The entire process of analysis/modification can be repeatedmany times to derive structural information while consuming only verysmall quantities of analyte (sometimes only a few femtomoles or less).The demonstrations of protein structure analysis based on AMS have todate included both N- and C-terminal sequence analyses and verificationof several types of sequence-specific posttranslational modificationsincluding phosphorylation and dephosphorylation, glycosylation, cysteineresidue reactivity, site-specific chemical modifications (e.g.,Histidine residues), and ligand binding.

Beyond biopolymer sequence determinations and the solution of individualbiopolymer structures, is the ability to understand the structuraldeterminants of functional supramolecular assemblies. The opportunity toinvestigate the structural determinants of higher order (e.g.,quaternary) structures is also presented by AMS. It has beendemonstrated by using the present invention that noncovalent molecularrecognition events, some not readily observed by more traditionalbioanalytical procedures (often requiring disruption of equilibrium andstructure dissociating conditions), are investigated directly by theevaluation of molecular associations (i.e., recognition) withmacromolecular analytes that have been tethered, directly or indirectly,to the probe element surface.

As used herein, "analyte" refers to any atom and/or molecule; includingtheir complexes and fragment ions. In the case of biologicalmacromolecules, including but not limited to: protein, peptides, DNA,RNA, carbohydrates, steroids, and lipids. Note that most importantbiomolecules under investigation for their involvement in the structureor regulation of life processes are quite large (typically severalthousand times larger than H₂ O).

As used herein, the term "molecular ions" refers to molecules in thecharged or ionized state, typically by the addition or loss of one ormore protons (H⁺).

As used herein, the term "molecular fragmentation" or "fragment ions"refers to breakdown products of analyte molecules caused, for example,during laser-induced desorption (especially in the absence of addedmatrix).

As used herein, the term "solid phase" refers to the condition of beingin the solid state, for example, on the probe element surface.

As used herein, "gas" or "vapor phase" refers to molecules in thegaseous state (i.e., in vacuo for mass spectrometry).

As used herein, the term "analyte desorption/ionization" refers to thetransition of analytes from the solid phase to the gas phase as ions.Note that the successful desorption/ionization of large, intactmolecular ions by laser desorption is relatively recent (circa1988)--the big breakthrough was the chance discovery of an appropriatematrix (nicotinic acid).

As used herein, the term "gas phase molecular ions" refers to those ionsthat enter into the gas phase. Note that large molecular mass ions suchas proteins (typical mass=60,000 to 70,000 times the mass of a singleproton) are typically not volatile (i.e., they do not normally enterinto the gas or vapor phase). However, in the procedure of the presentinvention, large molecular mass ions such as proteins do enter the gasor vapor phase.

As used herein in the case of MALDI, the term "matrix" refers to any oneof several small, acidic, light absorbing chemicals (e.g., nicotinic orsinapinic acid) that is mixed in solution with the analyte in such amanner so that, upon drying on the probe element, the crystallinematrix-embedded analyte molecules are successfully desorbed (by laserirradiation) and ionized from the solid phase (crystals) into thegaseous or vapor phase and accelerated as intact molecular ions. For theMALDI process to be successful, analyte is mixed with a freshly preparedsolution of the chemical matrix (e.g., 10,000:1 matrix:analyte) andplaced on the inert probe element surface to air dry just before themass spectrometric analysis. The large fold molar excess of matrix,present at concentrations near saturation, facilitates crystal formationand entrapment of analyte.

As used herein, "energy absorbing molecules (EAM)" refers to any one ofseveral small, light absorbing chemicals that, when presented on thesurface of a probe element (as in the case of SEND), facilitate the neatdesorption of molecules from the solid phase (i.e., surface) into thegaseous or vapor phase for subsequent acceleration as intact molecularions. The term EAM is preferred, especially in reference to SEND. Notethat analyte desorption by the SEND process is defined as asurface-dependent process (i.e., neat analyte is placed on a surfacecomposed of bound EAM). In contrast, MALDI is presently thought tofacilitate analyte desorption by a volcanic eruption-type process that"throws" the entire surface into the gas phase. Furthermore, note thatsome EAM when used as free chemicals to embed analyte molecules asdescribed for the MALDI process will not work (i.e., they do not promotemolecular desorption, thus they are not suitable matrix molecules).

As used herein, "probe element" or "sample presenting device" refers toan element having the following properties: it is inert (for example,typically stainless steel) and active (probe elements with surfacesenhanced to contain EAM and/or molecular capture devices).

As used herein, "MALDI" refers to Matrix-Assisted LaserDesorption/Ionization As used herein, "TOF" stands for Time-of-Flight.

As used herein, "MS" refers to Mass Spectrometry.

As used herein "MALDI-TOF MS" refers to Matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry.

As used herein, "ESI" is an abbreviation for Electrospray ionization.

As used herein, "chemical bonds" is used simply as an attempt todistinguish a rational, deliberate, and knowledgeable manipulation ofknown classes of chemical interactions from the poorly defmed kind ofgeneral adherence observed when one chemical substance (e.g., matrix) isplaced on another substance (e.g., an inert probe element surface).Types of defined chemical bonds include electrostatic or ionic (+/-)bonds (e.g., between a positively and negatively charged groups on aprotein surface), covalent bonds (very strong or "permanent" bondsresulting from true electron sharing), coordinate covalent bonds (e.g.,between electron donor groups in proteins and transition metal ions suchas copper or iron), and hydrophobic interactions (such as between twononcharged groups).

As used herein, "electron donor groups" refers to the case ofbiochemistry, where atoms in biomolecules (e.g, N, S, O) "donate" orshare electrons with electron poor groups (e.g., Cu ions and othertransition metal ions).

The present invention uses a general category of probe elements (i.e.,sample presenting means) with Surfaces Enhanced for LaserDesorption/Ionization (SELDI), within which there are three (3) separatesubcategories. Surfaces Enhanced for Neat Desorption (SEND) where theprobe element surfaces (i.e., sample presenting means) are designed tocontain Energy Absorbing Molecules (EAM) instead of "matrix" tofacilitate desorption/ionizations of analytes added directly (neat) tothe surface. Note that this category 1 (SEND) is used alone or incombination with Surfaces Enhanced for Affinity Capture (SEAC)(category2), where the probe element surfaces (i.e., sample presenting means) aredesigned to contain chemically defined and/or biologically definedaffinity capture devices to facilitate either the specific ornonspecific attachment or adsorption (so-called docking or tethering) ofanalytes to the probe surface, by a variety of mechanisms (mostlynoncovalent). Note that category 2 (SEAC) is used with added matrix orit is used in combination with category 1 (SEND) without added matrix.Thus, the combination of SEND and SEAC actually represents a distinctivecategory.

Category 3 involves Surfaces Enhanced for Photolabile Attachment andRelease (SEPAR) where the probe element surfaces (i.e., samplepresenting means) are designed/modified to contain one or more types ofchemically defined crosslinking molecules to serve as covalent dockingdevices. These Photolabile Attachment Molecules (PAM) are bivalent ormultivalent in character, that is, one side is first reacted so as topermanently attach the PAM to the probe element surface of the samplepresenting means, then the other reactive side(s) of the PAM is ready tobe reacted with the analyte when the analyte makes contact with thePAM-derivatized probe surface. Such surfaces (i.e., sample presentingmeans) allow for very strong (i.e., stable, covalent) analyte attachmentor adsorption (i.e., docking or tethering) processes that are covalentbut reversible upon irradiation (i.e., photolabile). Such surfacesrepresent platforms for the laser-dependent desorption of analytes thatare to be chemically and/or enzymatically modified in situ (i.e.,directly on the probe tip) for the purpose of structure elucidation.Only those analytes on the probe surface that are actually irradiated(small percentage of total) is desorbed. The remainder of the tetheredanalytes remain covalently bound and is modified without loss due tosome inadvertent uncoupling from the surface. Note that the SEPARcategory (category 3) is characterized by analyte attachment processesthat are reversible upon exposure to light. However, the light-dependantreversal of the analyte surface attachment bond(s) does not necessarilyenable analyte desorption into the gas phase per se. In other words, themolecules responsible for the photolabile attachment of the analytes tothe probe surface are not necessarily the same as the Energy AbsorbingMolecules (EAM) described for SEND. But here is an important exception:The present invention includes some hybrid EAM/PAM chemicals that havedual functionality with respect to SEND and SEPAR. That is, some EAMmolecules presently used for SEND can be modified to act as mediators ofboth the SEND and SEPAR processes. Similarly, some hybrid affinitycapture/PAM chemicals that have dual functionality with respect to SEACand SEPAR are provided. The present invention uses some affinity capturedevices, particularly those that are biologically defined, that aremodified to act as mediators of both the SEAC and SEPAR processes.

The invention herein presents, a sample presenting means (i.e., probeelement surface) with surface-associated (or surface-bound) molecules topromote the attachment (tethering or anchoring) and subsequentdetachment of tethered analyte molecules in a light-dependent manner,wherein the said surface molecule(s) are selected from the groupconsisting of photoactive (photolabile) molecules that participate inthe binding (docking, tethering, or crosslinking) of the analytemolecules to the sample presenting means (by covalent attachmentmechanisms or otherwise). Further, a sample presenting means (composedof one or more of the suitable probe element materials described inprevious claims), wherein analyte(s) are bound to the surface saidsample presenting means by one or more photolabile bonds so thatincident pulse(s) of light (e.g., from one or more lasers) is used tobreak the photolabile bond(s) tethering the analyte(s) to the probeelement surface in a manner that is consistent with the subsequentdesorption of the analyte from the stationary (solid) phase surface ofthe probe into the gas (vapor) phase is also presented.

The chemical specificity(ies) determining the type and number of saidphotolabile molecule attachment points between the SEPAR samplepresenting means (i.e., probe element surface) and the analyte (e.g.,protein) may involve any one or more of a number of different residuesor chemical structures in the analyte (e.g., His, Lys, Arg, Tyr, Phe,and Cys residues in the case of proteins and peptides). In other words,in the case of proteins and peptides, the SEPAR sample presenting meansmay include probe surfaces modified with several different types ofphotolabile attachment molecules to secure the analyte(s) with aplurality of different types of attachment points.

The wavelength of light or light intensity (or incident angle) requiredto break the photolabile attachment(s) between the analyte and the probeelement surface may be the same or different from the wavelength oflight or light intensity (or incident angle) required to promote thedesorption of the analyte from the stationary phase into the gas orvapor phase.

The photolabile attachment of the analyte(s) to the probe elementsurface (i.e., sample presenting means), particularly biopolymers suchas peptides, proteins, ribonucleic acid (RNA), deoxyribonucleic acids(DNA), and carbohydrates (CHO), may involve multiple points ofattachment between the probe surface and the analyte macromolecule. Oncethe biopolymer is attached via multiple points of attachment, differentpoints in the backbone of the biopolymer may be deliberately cut orfragmented by chemical and/or enzymatic means so that many of theresulting fragments are now separate and distinct analytes, each onestill attached (tethered) to the probe surface by one or morephotolabile bonds, to be desorbed into the gas phase in parallel forsimultaneous mass analyses with a time-of-flight mass analyzer. Thisprocess enables biopolymer (protein, peptides, RNA, DNA, carbohydrate)sequence determinations to be made.

As used herein "affinity" refers to physical and/or chemical attractionbetween two molecules. Typically used in nature for purposes ofstructure or regulation of bioactivity (i.e., information transfer).Usually the affinity of one biomolecule for another is quite specific.Used in the present case to describe principle by which molecularanalytes of interest are captured. In the case of SEAC, chemicals orbiomolecules with a characteristic affinity for the analyte(s) ofinterest are tethered (bound) to the surface of the probe element toactively "seek" out and selectively bind the desired analyte.

As used herein, "molecular recognition" refers to the interaction eventbetween two molecules with a natural affinity for one another.

As used herein, "molecular capture" refers to the use of tetheredbiomolecules to attract and bind (capture) other biomolecules for whicha specific affinity relationship exists.

As used herein, "passive adsorption" refers to the act of simply placingthe analyte (e.g., with matrix).

As used herein, "active docking" refers to the deliberate capture ofanalyte molecules on the surface of an active probe element as in thecase of SEAC.

As referred to herein "stationary phase" means the same as solid phase.In the present context either the probe element surface itself or one ofthe "external" particulate SEND or SEAC devices used in conjunction withan inert probe element surface.

As used herein, "active surface area" refers to that area of the surfacethought or known to participate in the desired reaction or event (e.g.,EAM attachment or affinity capture). The active surface area may besignificantly less than the total surface area (due to physical effectssuch as steric hinderance, some of the total area may not be availableor useful).

As used herein, "ligand" refers to a typically relatively small molecule(bait) that binds to a large biomolecule (fish). In the present case,ligands are attached (chemically bound) through a linker arm (fishingline) to the probe element surface. This process allows the biomolecularcapture event to be localized on the surface (stationary or solidphase).

As used herein, "affinity reagent" refers to an analyte capture device,viz., the class of molecules (both man made, unnatural, natural andbiological) and/or compounds which have the ability of being retained onthe presenting surface (by covalent bonding, chemical absorption, etc.)while retaining the ability of recognition and bonding to an analyte.

As used herein, "desorption" refers to the departure of analyte from thesurface and/or the entry of the analyte into a gaseous phase.

As used herein, "ionization" refers to the process of creating orretaining on an analyte an electrical charge equal to plus or minus oneor more electron units.

As used herein, "adduct" refers to the appearance of an additional massassociated with the analyte and usually caused by the reaction of excessmatrix (or matrix break-down products) directly with the analyte.

As used herein, "adsorption"--the chemical bonding (covalent and/ornoncovalent) of the energy-absorbing molecules, the affinity reagent(i.e., analyte capture device), and/or the analyte to the probe(presenting surface).

One embodiment of the present invention is an apparatus for measuringthe mass of an analyte molecule of an analyte sample by means of massspectrometry, said apparatus comprising: a spectrometer tube; vacuummeans for applying a vacuum to the interior of said tube; electricalpotential means within the tube for applying an accelerating electricalpotential to desorbed analyte molecules from said analyte sample;

sample presenting means removably insertable into said spectrometertube, for presenting said analyte sample in association with surfaceassociated molecule for promoting desorption and ionization of saidanalyte molecules, wherein said surface molecule is selected from thegroup consisting of energy absorbing molecule, affinity capture device,photolabile attachment molecule and combination thereof; an analytesample deposited on said sample presenting means in association withsaid surface associated molecules;

whereby at least a portion of said analyte molecules not consumed insaid mass spectrometry analysis will remain accessible for subsequentchemical, biological or physical analytical procedures; laser beam meansfor producing a laser beam directed to said analyte sample for impartingsufficient energy to desorb and ionize a portion of said analytemolecules from said analyte sample; and detector means associated withsaid spectrometer tube for detecting the impact of accelerated ionizedanalyte molecules thereon.

Another embodiment of the present invention is a method in massspectrometry to measure the mass of an analyte molecule, said methodcomprising the steps of: derivitizing a sample presenting surface on aprobe tip face with an affinity capture device having means for bindingwith an analyte molecule; exposing said derivitized probe tip face to asource of said analyte molecule so as to bind said analyte moleculethereto; placing the derivitized probe tip with said analyte moleculesbound thereto into one end of a time-of-flight mass spectrometer andapplying a vacuum and an electric field to form an acceleratingpotential within the spectrometer; striking at least a portion of theanalyte molecules bound to said derivitized probe tip face within thespectrometer with one or more laser pulses in order to desorb ions ofsaid analyte molecules from said tip; detecting the mass of the ions bytheir time of flight within said mass spectrometer; and displaying suchdetected mass. In an preferred embodiment, this method further comprisesapplying a desorption/ionization assisting matrix material to said probetip face in association with said affinity capture device. In a morepreferred embodiment, the method according further comprises removingsaid probe tip from said mass spectrometer; performing a chemical orbiological procedure on said portion of said analyte molecules notdesorbed to alter the composition of said portion of said analytemolecules not desorbed; reinserting said probe tip with said alteredanalyte molecules thereon; and performing subsequent mass spectrometryanalysis to determine the molecular weight of said altered analytemolecules.

In an additional embodiment, said affinity capture device is chemicallybonded to said face of said probe tip, physically adhered to said faceof said probe tip, adapted to chemically bond to said analyte molecules,or adapted to biologically adhere to said analyte molecules. In afurther embodiment, said analyte molecules are biomolecules and saidaffinity reagent is adapted to selectively isolate said biomoleculesfrom an undifferentiated biological sample. In a preferred embodiment,said matrix materials are in the weakly acidic to strongly basic pHrange. In a more preferred embodiment, said matrix materials have a pHabove 6.0. Further, an additional embodiment presents the face of saidprobe tip formed of an electrically insulating material.

An additional embodiment of the present invention is a method ofmeasuring the mass of analyte molecules by means of laserdesorption/ionization, time-of-flight mass spectrometry in which anenergy absorbing material is used in conjunction with said analytemolecules for facilitating desorption and ionization of the analytemolecules, wherein the improvement comprises presenting the analytemolecules on or above the surface of the energy absorbing material,wherein at least a portion of the analyte molecules not desorbed in saidmass spectrometry analysis remain chemically accessible for subsequentanalytical procedures.

A further embodiment of the present invention is an apparatus forfacilitating desorption and ionization of analyte molecules, saidapparatus comprising: a sample presenting surface; and surfaceassociated molecules, wherein said surface associated molecules areselected from the group consisting of energy absorbing molecule,affinity capture device, photolabile attachment molecule and combinationthereof, said surface associated molecules associated with said samplepresenting surface and having means for binding with said analytemolecules.

In a preferred embodiment, said sample presenting surface comprises thesurface of a probe tip for use in a time-of-flight mass spectrometryanalyzer. In addition, the preferred embodiment presents an affinitycapture device or photolabile attachment molecule that is chemicallybonded to said sample presenting surface, physically adhered to saidsample presenting surface, chemically bonded to said analyte molecules,or is adapted to biologically adhere to said analyte molecules. Further,the preferred embodiment presents analyte molecules are biomolecules andsaid affinity capture device or photolabile attachment molecule isadapted to selectively isolate said biomolecules from anundifferentiated biological sample.

In addition, the apparatus may have a matrix material deposited on saidsample presenting surface in association with said affinity capturedevice or photolabile attachment molecule. In a more preferredembodiment, the matrix material is in the weakly acidic to stronglybasic pH range. In a most preferred embodiment, the matrix material hasa pH above 6.0. Additionally, a preferred embodiment includes a samplepresenting surface formed of an electrically insulating material.

In an additional embodiment of the present invention, there is presenteda method for capturing analyte molecules on a sample presenting surfaceand desorbing/ionizing said captured analyte molecules from said samplepresenting surface for subsequent analysis, said method comprising:derivitizing said sample presenting surface with an affmity capturedevice or photolabile attachment molecule having means for binding withsaid analyte molecules; exposing said derivitized sample present surfaceto a sample containing said analyte molecules; capturing said analytemolecules on said derivitized sample presenting surface by means of saidaffinity capture device or photolabile attachment molecule; and exposingsaid analyte molecules, while bound to said derivitized samplepresenting surface by means of said affinity capture device orphotolabile attachment molecule, to an energy or light source to desorbat least a portion of said analyte molecules from said surface.

A further embodiment of the present invention is a method for preparinga surface for presenting analyte molecules for analysis, said methodcomprising: providing a substrate on said surface for supporting saidanalyte; derivitizing said substrate with an affinity capture device orphotolabile attachment molecule having means for selectively bondingwith said analyte; and a means for detecting said analyte moleculesbonded with said affinity capture device or photolabile attachmentmolecule. In a preferred embodiment, there is provided the additionalstep of applying a detection material to said surface. In a morepreferred embodiment, such detection material comprises a fluorescingspecies, an enzymatic species, a radioactive species, or alight-emitting species.

In an additional preferred embodiment, the step of depositing adesorption/ionization assisting material to said sample presentingsurface in association with said affinity capture device or photolabileattachment molecule is included. In a further preferred embodiment, theenergy source comprises a laser. In another preferred embodiment, anaffinity capture device is used and said energy source comprises an ionsource. Further, a preferred embodiment may include a portion of saidanalyte molecules remaining bound to said sample presenting surfaceafter exposure to said energy source. In a more preferred embodiment,the additional steps of converting at least a portion of the analytemolecules remaining bound on said derivitized sample presenting surfaceto modified analyte molecules by a chemical, biological or physicalreaction, wherein said analyte molecules remain bound to saidderivitized sample presenting surface by means of said affinity capturedevice or photolabile attachment molecule; and exposing said modifiedanalyte molecules to an energy source so as to desorb at least a portionof said modified analyte molecules from said surface are included.

In an embodiment of the present invention, a sample probe for promotingdesorption of intact analytes into the gas phase comprising: a samplepresenting surface; and an energy absorbing molecule associated withsaid sample presenting surface, wherein said sample probe promotesdesorption of an intact analyte molecule positioned on, above or betweenthe energy absorbing molecules when said sample probe is impinged by anenergy source is provided. In a more preferred embodiment, the energyabsorbing molecule is selected from the group consisting of cinnamamide,cinnamyl bromide, 2,5-dihydroxybenzoic acid andα-cyano-4-hydroxycinnamic acid. Also in a preferred embodiment, one mayutilize a sample presenting surface selected from the group consistingof glass, ceramics, teflon coated magnetic materials; organic polymersand native biopolymers.

In another embodiment of the present invention, there is provided asample probe for promoting desorption of intact analytes into the gasphase comprising: a sample presenting surface; and an affinity capturedevice associated with said sample presenting surface; wherein, whensaid sample probe is impinged by an energy source, said sample probepromotes the transition of an intact analyte molecule into the gasphase. In a preferred embodiment, the affinity capture device isselected from the group consisting of metal ions, proteins, peptides,enzymes immunoglobulins, nucleic acids, carbohydrates, lectins, dyes,reducing agents and combination thereof. In another preferredembodiment, the sample presenting surface is selected from the groupconsisting of glass, ceramics, teflon coated magnetic materials; organicpolymers and native biopolymers.

An additional embodiment presents a sample probe for desorption ofintact analyte into the gas phase, comprising: a sample presentationsurface; and a surface associated molecule wherein said surfaceassociated molecule is a photolabile attachment molecule having at leasttwo binding sites, wherein at least one site is bound to the samplepresentation surface and at least one site is available to bind ananalyte and wherein the analyte binding site is photolabile.

In another embodiment, there is provided a sample probe for promotingdesorption of intact analytes into the gas phase comprising: a samplepresentation surface; and either a mixture of at least two differentmolecules selected from the group consisting of an affinity capturedevice, an energy absorbing molecule and a photolabile attachmentmolecule associated with said sample presentation surface; wherein whenan analyte is associated with said sample probe, said sample probepromotes the transition of the analyte into the gas phase when saidsample probe is impinged by an energy source; or at least two differentaffinity capture devices associated with said sample presentationsurface; wherein, when said sample probe is impinged by an energysource, said sample probe promotes the transition of an analyte moleculeinto the gas phase at different rates depending on the affinity capturedevice associated with said analyte molecule.

In a preferred embodiment, the analyte is selectively desorbed from themixture after impingement by the energy source. In another preferredembodiment, the affinity devices are arranged in predetermined arrays.This can be accomplished by arranging the affinity adsorption "spots"(0.005 to 0.080 inch diameter) on the probe surface in a defined manner(400 to 1,000 spots could be placed on a surface about the size of aglass slide). In a more preferred embodiment, the arrays selectivelyabsorb a plurality of different analytes.

In a more preferred embodiment, an apparatus of the present invention isused to quantitate an analyte, wherein the position and quantity ofaffinity capture devices determines the quantity of analyte absorbed. Inanother preferred embodiment, the binding may be selective ornon-selective.

In an additional embodiment, a sample probe for promoting desorption ofintact analyte into the gas phase, comprising: a sample presentationsurface; and either a surface associated molecule, wherein said surfaceassociated molecule can function both as an energy absorbing moleculeand as an affinity capture device; or a surface associated moleculewherein said surface associated molecule is a photolabile attachmentmolecule having at least two binding sites, wherein at least one site isbound to the sample presentation surface and at least one site isavailable to bind an analyte and wherein the analyte binding site isphotolabile.

A different embodiment of the present invention includes a method inmass spectrometry to measure the mass of an analyte molecule, saidmethod comprising the steps of: derivitizing a sample presenting surfaceon a probe tip face with a photolabile attachment molecule (PAM),wherein said PAM has at least two binding sites, one binding site bindsto the sample presenting surface and at least one binding site isavailable for binding with an analyte molecule; exposing saidderivitized probe tip face to a source of said analyte molecule so as tobind said analyte molecule thereto; placing the derivitized probe tipwith said analyte molecules bound thereto into one end of atime-of-flight mass spectrometer and applying a vacuum and an electricfield to form an accelerating potential within the spectrometer;striking at least a portion of the analyte molecules bound to saidderivitized probe tip face within the spectrometer with one or morelaser pulses in order to desorb ions of said analyte molecules from saidtip; detecting the mass of the ions by their time of flight within saidmass spectrometer; and displaying such detected mass. In a preferredembodiment, the step of applying a desorption/ionization assistingmatrix material to said probe tip face in association with said PAM isincluded. In a more preferred embodiment, an additional steps ofremoving said probe tip from said mass spectrometer; performing achemical, biological or physical procedure on said portion of saidanalyte molecules not desorbed to alter the composition of said portionof said analyte molecules not desorbed; reinserting said probe tip withsaid altered analyte molecules thereon; and

performing subsequent mass spectrometry analysis to determine themolecular weight of said altered analyte molecules are included. Apreferred embodiment may also include PAM being chemically bonded tosaid face of said probe tip; PAM being chemically bonded to said analytemolecule, wherein said bond between the PAM and the analyte molecule isbroken and the analyte molecule is released in a light dependent manner;or, where said analyte molecules are biomolecules, said PAM is adaptedto selectively isolate said biomolecules from an undifferentiatedbiological sample. In another preferred embodiment, said matrixmaterials are in the weakly acidic to strongly basic pH range. In a morepreferred embodiment, said matrix materials have a pH above 6.0. Apreferred embodiment may also include the face of said probe tip beingformed of an electrically insulating material.

A further embodiment presents a method of measuring the mass of analytemolecules by means of laser desorption/ionization, time-of-flight massspectrometry in which a photolabile attachment molecule (PAM) is used inconjunction with said analyte molecules for facilitating desorption andionization of the analyte molecules, the improvement comprising:presenting the analyte molecules on or above the surface of the PAM,wherein at least a portion of the analyte molecules not desorbed in saidmass spectrometry analysis remain chemically accessible for subsequentanalytical procedures.

Another embodiment of the present invention is a sample probe forpromoting of differential desorption of intact analyte into the gasphase, comprising: a sample presentation surface; and at least twodifferent photolabile attachment molecules associated with said samplepresentation surface; wherein, when said sample probe is impinged by anenergy source, said sample probe promotes the transition of an analytemolecule into the gas phase at different rates depending on thephotolabile attachment molecule associated with said analyte molecule.In a preferred embodiment, the photolabile attachment molecules arearranged in predetermined arrays. In a more preferred embodiment, thearrays selectively absorb a plurality of different analytes.

An additional embodiment of the present invention includes a sampleprobe for promoting desorption of intact analytes into the gas phasecomprising: a sample presenting surface; and a photolabile attachmentmolecule associated with said sample presenting surface; wherein, whensaid sample probe is impinged by an energy source, said sample probepromotes the transition of an intact analyte molecule into the gasphase. In a preferred embodiment, and analyte is quantitated, whereinthe position and quantity of photolabile attachment molecule determinesthe quantity of analyte absorbed.

Another embodiment shows a method for biopolymer sequence determinationcomprising the steps of: binding a biopolymer analyte to probe tipcontaining a sample presenting surface having a surface selectedmolecule selected from the group consisting of an energy absorbingmolecule, an affinity capture device, a photolabile attachment moleculeand a combination thereof; desorption of biopolymer analyte in massspectrometry analysis, wherein at least a portion of said biopolymer isnot desorbed from the probe tip; analyzing the results of the desorptionmodifying the biopolymer analyte still bound to the probe tip; andrepeating the desorption, analyzing and modifying steps until thebiopolymer is sequenced. A preferred embodiment presents the biopolymerselected from the group consisting of protein, RNA, DNA andcarbohydrate.

The following specific examples describe specific embodiments of thepresent invention and its materials and methods, are illustrative of theinvention and are not intended to limit the scope of the invention.

The examples of the present invention utilize a time-of-flight massspectrometer with a high energy source, such as a laser beam, tovaporize the analyte from the surface of a probe tip. In the process,some of the molecules are ionized. The positively charged molecules arethen accelerated through a short high voltage field and enter into afield-free flight tube. A sensitive detector positioned at the end ofthe flight tube gives a signal as each molecular ion strikes it. Oneskilled in the art recognizes that other modes of detection andionization can also be used.

EXAMPLE 1 Energy Absorbing Molecules in Aqueous, Neutralized Form

Prior art matrix material used in matrix-assisted laser desorptiontime-of-flight mass spectrometry are strongly acidic. One of the presentdiscoveries is that analytes is desorbed when mixed with neutralizedenergy absorbing molecules dissolved in entirely aqueous solvents. Bysuitable neutralization to pH 6.0 or above, the matrix material is madelargely passive to subsequent chemical or enzymatic reactions carriedout on the analyte molecules presented on the probe tip surfaces. Sinceonly a small fraction of the analyte molecules are used in eachdesorption/mass spectrometer measurement, the samples on the probe tipsare available for in situ sequential chemical or enzymaticmodifications. After modification the samples are analyzed by massspectrometry. Analysis on the same probe tips provides a more accuratedetermination of the molecule and its characteristics, including itsstructure.

Mass spectrometry is performed on a modified Vestec model VT2000 or aMAS model SELDI Research Linear time-of-flight mass spectrometer whichuses a frequency-tripled output from a Q-switched neodymiumyttriumaluminum garnet (Nd-YAG) pulsed laser (355 nm, 5 ns pulse). Ionsdesorbed by pulsed laser irradiation are accelerated to an energy of 30keV and allowed to drift along a 2-meter field free drift region(maintained at 10⁻⁸ torr). Ion signals detected using a 20-stagediscrete dynode electron multiplier are amplified by a factor of 10using a fast preamplifier prior to being recorded using a 200 MS/stransient recorder (LeCroy TR8828D, 8-bit y-axis resolution) or aTektronix digitizer capable of fast signal averaging. The lasterirradiance is adjusted real-time, while monitoring the process on anoscilloscope (Tektronix), in order to achieve optimum ion signal. Datareduction (peak centroid calculations and time to mass/chargeconversions) are performed with PC-based software. A VG TOFSpec massspectrometer which uses a nitrogen laser generating pulsed laser at 335nm. or a Linear LDI 1700 mass spectrometer which uses a nitrogen lasergenerating pulsed laser 335 nm. may also be used.

I. Specific Analysis

1. Sinapinic acid (Aldrich Chemical Co., Inc., Milwaukee, Wis.) issuspended in water at 20 mg/ml (pH 3.88) and neutralized withtriethylamine (Pierce, Rockford, Ill.) to pH 6.2-6.5. An aqueous mixture(1 μl) of synthetic peptides, containing human histidine richglycoprotein metal-binding domains (GHHPH)₂ G (1206 Da), (GHHPH)₅ G(2904 Da), and human estrogen receptor dimerization domain (D473-L525)(6168.4 Da) is mixed with 2 μl sinapinic acid (20 mg/ml water, pH 6.2)on a probe tip and analyzed by laser desorption time-of-flight massspectrometry. After acquiring five spectra (average 100 laser shots perspectrum), the probe is retrieved, 2 μl of 20 mM Cu(SO)₄ is added andthe sample is reanalyzed by mass spectrometry. FIG. 1 (upper profile)shows the mass spectrum of the three peptides desorbed in the presenceof neutralized energy absorbing molecules. FIG. 1 (lower profile) showsthe in situ metal-binding of the peptides in the presence of neutralenergy absorbing molecules. The (GHHPH)₂ G peptide can bind at least 4Cu(II), the (GHHPH)₅ G peptide can bind at least 5 Cu(II) and thedimerization domain can bind at least 1 Cu(II) under the presentexperimental conditions. Similar result is obtained withα-cyano-4-hydroxycinnamic acid (20 mg/ml water) neutralized to pH 6.5.

2. An aliquot of 1 μl of human β casein phosphopeptide (R1-K18+5P) (2488Da) is mixed with 1 μl of sinapinic acid (20 mg/ml water) neutralized topH 6.5, and analyzed by laser desorption time-of-flight massspectrometry. After acquiring five spectra (average 100 laser shots perspectrum), the probe is removed, the remaining phosphopeptide mixed withthe neutralized sinapinic acid is digested directly on the probe tip by0.5 μl of alkaline phosphatase (Sigma) and incubated at 23° C. for 5min. After acquiring five spectra (average 100 laser shots perspectrum), the probe is removed, further digestion on remainingphosphopeptides is carried out by adding another aliquot of 0.5 μl ofalkaline phosphatase and incubated at 23° C. for 5 min. The sample isre-analyzed by laser desorption mass spectrometry. FIG. 2 (top profile)shows the mass spectrum of the phosphopeptide desorbed in the presenceof neutralized energy absorbing molecules. FIG. 2 (second from topprofile) shows the in situ 5 min alkaline phosphatase digestion toremove phosphate groups from the phosphopeptide. The 0P, 1P and 3P peaksrepresent the products after removal of five, four and two phosphategroups respectively from the phosphopeptide. FIG. 2 (third from topprofile) shows that further in situ digestion with alkaline phosphatasecan result in almost complete removal of all phosphate groups from thephosphopeptide. In contrast, FIG. 2 (bottom profile) shows that in thecontrol experiment where in situ alkaline phosphatase (0.5 μl) digestionis carried out in the presence of energy absorbing molecules withoutprior neutralization (e.g. sinapinic acid at pH 3.88 or dihydroxybenzoicacid at pH 2.07), very limited digestion occurred in 10 min at 23° C.

3. An aliquot of 1 μl of (GHHPH)₅ G peptide (2904 Da) is mixed with 2 μlof sinapinic acid (20 mg/ml water) neutralized to pH 6.2, and analyzedby laser desorption time-of-flight mass spectrometry. After acquiringfive spectra (average 100 laser shotsper spectrum), the remainingpeptides mixed with neutralized sinapinic acid are digested directly onthe probe tip by 1 μl of carboxypeptidase P (Boehringer Mannheim Corp,Indianapolis, Ind.) and incubated at 23° C. for 30 min. The sample isanalyzed by mass spectrometry. FIG. 3 shows a composite mass spectra ofthe peptide before (lower profile) and after (upper profile) in situdigestion by carboxypeptidase P in the presence of neutralized energyabsorbing molecules. The decrease in mass represents the removal of aGly residue from the C-terminal of the peptide.

These examples illustrate that neutralized energy absorbing molecules inaqueous solutions are more biocompatible in preserving the structure andfunction of the analytes even when added in large molar excess. Theirpresence results in no interference to in situ sequential chemical orenzymatic reactions on the remaining analyte.

EXAMPLE 2 Nonmetallic Probe Elements (Sample Presenting Surfaces)

It has been found that the probe elements (probe tips or samplepresenting surfaces) used in the process of the invention need not bemetal or metal-coated, as described in prior art procedures. The samplepresenting surfaces are composed of a variety of materials, includingporous or nonporous materials, with the porous materials providingsponge-like, polymeric, high surface areas for optimized adsorption andpresentation of analyte.

Polypropylene or polystyrene or polyethylene or polycarbonate are meltedin an open flame and deposited as a thin layer on a 2 mm diameterstainless steel probe element so as to cover it completely. Solid glassrod or solid nylon filaments (up to 1.5 mm diameter) or polyacrylamiderod are cut into 1 cm segments and inserted into the stainless steelprobe support. Magnetic stir bars (1.5×8 mm, teflon-coated) are insertedinto stainless steel probe tip support. An aliquot of 1 μl of peptidemixture containing (GHHPH)₅ G and human estrogen receptor dimerizationdomain, is mixed with 2 μl of dihydroxybenzoic acid (dissolved in 30%methanol, 0.1% trifluoroacetic acid) on each of such probe elements andanalyzed by laser desorption time-of-flight mass spectrometry. FIG. 4shows that analytes could be desorbed from several examples ofinsulating, biocompatible surfaces.

These surfaces can be derivatized (at varying densities) to bind bychemical bonds (covalent or noncovalent) affinity adsorption reagents(affinity capture devices), energy absorbing molecules (bound"matrix"molecules) or photolabile attachment molecules. The geometry ofthe sample presenting surface is varied (i.e., size, texture,flexibility, thickness, etc.) to suit the need (e.g., insertion into aliving organism through spaces of predetermined sizes) of the experiment(assay).

EXAMPLE 3 Affinity-Directed Laser Desorption Surface Enhanced AffinityCapture, SEAC

This example describes the use of existing and new solid phase affinityreagents designed for the (1) capture (adsorption) of one or moreanalytes, (2) the preparation of these captured analytes (e.g., washingwith water or other buffered or nonbuffered solutions to removecontaminants such as salts, and multiple cycles of washing, such as withpolar organic solvent, detergent-dissolving solvent, dilute acid, dilutebase or urea), and (3) most importantly, the direct transfer of thesecaptured and prepared analytes to the probe surface for subsequentanalyte desorption (for detection, quantification and/or mass analysis).Affinity capture devices are immobilized on a variety of materials,including electrically insulating materials (porous and nonporous),flexible or nonrigid materials, optically transparent materials (e.g.,glass, including glass of varying densities, thicknesses, colors andwith varying refractive indices), as well as less reactive, morebiocompatible materials (e.g., biopolymers such as agarose, dextran,cellulose, starches, peptides, and fragments of proteins and of nucleicacids). The preferred probe tip, or sample surface, for selectiveadsorption/presentation of sample for mass analysis are (1) stainlesssteel (or other metal) with a synthetic polymer coating (e.g.,cross-linked dextran or agarose, nylon, polyethylene, polystyrene)suitable for covalent attachment of specific biomolecules or othernonbiological affinity reagents, (2) glass or ceramic, and/or (3)plastics (synthetic polymer). The chemical structures involved in theselective immobilization of affinity reagents to these probe surfaceswill encompass the known variety of oxygen-dependent, carbon-dependent,sulfur-dependent, and/or nitrogen-dependent means of covalent ornoncovalent immobilization.

I. Surface Immobilized Metal Ion as the Affnity Capture Device

1. Cu(II) ion is chelated by iminodiacetate (IDA) group covalentlyattached to either porous agarose beads (Chelating Sepharose Fast Flow,Pharmacia Biotech Inc., Piscataway, N.J., ligand density 22-30 μmole/mlgel) or solid silica gel beads (Chelating TSK-SW, ToyoSoda, Japan,ligand density 15-20 μmole/ml gel). A mixture of synthetic peptidescontaining neurotensin (1655 Da), sperm activating peptide (933 Da) andangiotensin I (1296.5 Da), is mixed with 50 μl packed volume of TSK-SWIDA-Cu(II) at pH 7.0 (20 mM sodium phosphate, 0.5M sodium chloride) at23° C. for 10 min. The gel is separated from the remaining peptidesolution by centrifugation and is then washed with 200 μl sodiumphosphate, sodium chloride buffer, pH 7.0 three times to removenonspecifically bound peptides. Finally, the gel is suspended in 50 μlof water. Aliquots of 2 μl gel suspension and nonadsorbed peptidesolution are mixed with 1 μl of sinapinic acid (dissolved in methanol)on a stainless steel probe tip and analyzed by laser desorptiontime-of-flight mass spectrometry. After acquiring five spectra (averageof 100 laser shots per spectrum) on various spots of the probe tip, thesinapinic acid is removed by methanol. An aliquot of 2 μl of 20 mM CuSO₄is added, then mixed with 1 μl of sinapinic acid and reanalyzed by laserdesorption time-of-flight mass spectrometry. After acquiring anotherfive spectra (average of 100 laser shots per spectrum) on various spotsof the probe tip, the sinapinic acid is removed by methanol. Theremaining peptide adsorbed on IDA-Cu(II) gel beads is then digested with1 μl of trypsin (Sigma) in 0.1M sodium bicarbonate, pH 8.0 at 23° C. for10 min in a moist chamber. The gel beads are then washed with water toremove enzyme and salt before 1 μl of sinapinic acid is added and thesample analyzed by laser desorption time-of-flight mass spectrometry.FIG. 5A, top profile, shows the molecular ions (and multiple Na-adducts)of sperm activating factor (933 Da) and neurotensin (1655 Da) in theremaining peptide solution unabsorbed by the IDA-Cu(II). There is nosignificant peak corresponding to angiotensin I (1296.5 Da). The massspectrum in FIG. 5A, middle profile, shows the angiotensin I plusNa-adduct peaks that are selectively adsorbed on the IDA-Cu(II) gel.When the IDA-Cu(II) gel is further washed with 500 μl of water twotimes, the resulting mass spectrum shows only the parent angiotensin Iion and no other adduct peaks (FIGS. 5A and 5B, bottom profiles, FIG.5B, middle profile, shows the in situ copper binding (1 and 2 Cu) by theangiotensin peptide. FIG. 5B, top profile, shows the in situ trypsindigestion of the angiotensin peptide at the single Arg2 position in thesequence.

This example illustrates that: a) laser desorption is successfullycarried out on analyte affinity adsorbed on surface-immobilized metalion; b) once bound, the surface is washed with various solvents toremove all contaminating compounds in the sample to give a very cleanmass spectrum of the analyte; c) the affinity capture device selectsonly the analyte of defined structure (in this case angiotensin I isselectively adsorbed from the peptide mixture by IDA-Cu(II) because thispeptide has a free N-terminal and two histidine amino acid residues inthe sequence, both properties are required for strong Cu(II)-binding;whereas both sperm activating factor and neurotensin have blockedN-terminal and no histidine amino acid residues in their sequences); d)structure and function analyses through sequential in situ chemical orenzymatic modifications is carried out on the adsorbed analyte withminimal loss at each step of reaction and wash; and e) a probe elementwith surface bound substrate (e.g., angiotensin I) is used to monitorspecific enzyme activity (e.g., trypsin) in situ (e.g., inside thegastrointestinal tract of the human body).

2. A solution of horse heart myoglobin (325 pmole, 16,952 Da) is mixedwith 50 μl of TSK-SW IDA-Cu(II) at pH 7.0 (20 mM sodium phosphate, 0.5Msodium chloride) at 23° C. for 10 min. The gel is separated from thesolution by centrifugation and then washed with 500 μl of buffer twotimes and 500 μl of water two times. The quantity of remaining myoglobinin all these solutions are then estimated spectrophotometrically, thequantity adsorbed on the gel can then be calculated. The gel issuspended in 50 μl of water and then serially diluted into water. Analiquot of 0.5 μl of the diluted gel suspension is mixed with 1 μl ofsinapinic acid (dissolved in 30% methanol, 0.1% trifluoroacetic acid)and analyzed by laser desorption time-of-flight mass spectrometry. FIG.6 shows that a detectable signal (signal/noise=6, after averaging 50laser shots) of myoglobin is obtained with a calculated quantity of 4 to8 fmole deposited on the probe tip.

This example illustrates that affinity adsorbed analytes on a surfaceare much more easier to transfer and are free from any loss bynonspecific adsorption to container and transfer device surfaces. Theadsorbed analyte is sequestered on predetermined areas (that are evenless than the laser spot size) of the sample presenting surface in low(atto to femtomole) quantities at a defined surface density or localconcentration required for the efficient detection by laserdesorption/ionization time-of-flight mass spectrometry.

3. The human β casein peptides (E2-K18) are synthesized on an AppliedBiosystem Model 430A Peptide Synthesizer using the NMP-HOBt protocol.The Ser residues to be phosphorylated are coupled to the peptide chainwithout side chain protecting group. The unprotected Ser are firstphosphinylated using di-t-butyl-N,N,-diisopropyl-phosphoramidite. Thephosphite ester is then oxidized with t-butyl peroxide, washed, andcleaved from the resin. All the side chain protecting groups are removedwith 95% trifluoroacetic acid. The crude phosphopeptides are extractedwith methyl tbutyl ether and dried. This crude preparation of syntheticphosphopeptides is dissolved in 50 mM MES, 0.15M sodium chloride, pH 6.5and mixed with 50 μl of tris(carboxymethyl)-ethylenediamine(TED)-Fe(III) immobilized on porous Sepharose (synthesized as describedby Yip, T.-T. and Hutchens, T. W., Protein Expression and Purification2: 355-362 (1991), ligand density 65 μmole/ml) at 23° C. for 15 min. Thegel is washed with 500 μl of the same buffer three times and then with500 μl of water once. An aliquot of 1 μl of gel is mixed with 1 μl ofsinapinic acid (dissolved in 30% methanol, 0.1% trifluoroacetic acid) onthe probe tip and analyzed by laser desorption time-of-flight massspectrometry. After acquiring five spectra (average of 100 laser shotsper spectrum) on various spots of the probe tip, the sinapinic acid isremoved by methanol, and the remaining phosphopeptides adsorbed onTED-Fe(III) is digested directly on the probe tip by 1 μl of alkalinephosphatase (ammonium sulfate suspension, Sigma) in 50 mM HEPES pH 7.0at 23° C. for 10 min. in a moist chamber. The gel is washed with waterto remove enzyme and salt. Sinapinic acid is added and the sample isreanalyzed by laser desorption time-of-flight mass spectrometry. FIG. 7(top profile) shows the distribution of casein peptide (1934 Da) withmultiple phosphorylated forms. After in situ alkaline phosphatasedigestion, only the original nonphosphorylated form remains (lowerprofile).

This example illustrates the application of SEAC as a quick monitor ofphosphopeptide synthesis in a crude mixture without prior cleanup. Theidentity of the phosphopeptide is readily confirmed by in situ alkalinephosphatase digestion.

4. Aliquots of 100 μl of preterm infant formula (SIMILAC, Meade Johnson)and gastric content of preterm infant aspirated 90 min after feeding ofthe formula are mixed with 50 μl of TED-Fe(III) Sepharose in 0.1M MES,0.15M sodium chloride, pH 6.5 at 23° C. for 15 min. The gel is washedwith 500 μl of the same buffer three times and then with 500 μl of wateronce. Aliquots of 1 μl of gel suspensions or preterm infant formula orgastric aspirate are mixed with 2 μl of sinapinic acid (dissolved in 50%acetonitrile, 0.1% trifluoroacetic acid) on the probe tip and analyzedby laser desorption time-of-flight mass spectrometry. FIG. 8 shows thatthe mass spectrum of whole gastric aspirate (second from top profile) isquite similar to that of whole infant formula (bottom profile) in the1,000-15,000 Da region. However, the mass spectra of analytesselectively adsorbed by TED-Fe(III) from the two samples are quitedifferent, there are more low molecular weight phosphopeptides (i.e.,bound by TED-Fe(III)) present in the gastric aspirate (top profile) thanin the formula (second from bottom profile) due to the gastricproteolytic digestion of phosphoproteins present in the formula.

This example illustrates that SEAC is particularly useful in analyzingspecific analytes in biological samples. Phosphopeptides are moredifficult to detect in the presence of other contaminating components ina complex sample because they are less ionized in the positive ion mode.However, when the phosphopeptides are selectively adsorbed and all othercomponents in the sample are removed, no such signal depression occurs.

5. Aliquots of 200 μl of human and bovine histidine-rich glycoproteinare mixed with 50 μl of IDA-Cu(II) Sepharose (Pharmacia) at pH 7.0 (20mM sodium phosphate, 0.5M sodium chloride) at 23° C. for 10 min. The gelis washed with 500 μl buffer two times and 500 μl water once. Aliquotsof 1 μl of gel are mixed with 2 μl of sinapinic acid (dissolved in 30%methanol 0.1% trifluoroacetic acid) and analyzed by laser desorptiontime-of-flight mass spectrometry. After acquiring five spectra (averageof 100 laser shots per spectrum) on various spots of the probe tip, thesinapinic acid is removed by methanol wash. The remaining glycoproteinsadsorbed on the IDA-Cu(II) gel is then digested with N-glycanase in 20mM sodium phosphate, 0.5M sodium chloride, 3M urea, pH 7.0 at 37° C.overnight in a moist chamber. After washing with water to remove enzymeand salt, 2 μl of sinapinic acid is added and the sample is analyzed bymass spectrometry. After acquiring five spectra (average of 100 lasershots per spectrum) on various spots of the probe tip, the sinapinicacid is removed by methanol. Aliquots of 2 μl of trypsin in 0.1M sodiumbicarbonate are added and incubated at 37° C. for 30 min in a moistchamber. After a water wash to remove enzyme and salt, sinapinic acid isadded and the sample is analyzed by mass spectrometry. After acquiringfive spectra (average of 100 laser shots per spectrum) on various spotsof the probe tip, the sinapinic acid is removed by methanol. Aliquots of2 μl of 20 mM CuSO₄ is added. This is followed by addition of 2 μl ofsinapinic acid and then analyses by mass spectrometry. After acquiringfive spectra (average of 100 laser shots per spectrum) on various spotsof the probe tip, the sinapinic acid is removed by methanol. Aliquots of2 μl of diethylpyrocarbonate (Sigma) in 5 mM HEPES, pH 6.5 are added andincubated at 23° C. for 30 min. After a water wash to remove chemicalsand buffer salts, 2 μl of sinapinic acid is added and the sample isanalyzed by mass spectrometry. To obtain a partial sequence of themetal-binding peptides, instead of modifying the histidine residues withdiethylpyrocarbonate, add 1 ul of carboxypeptidase Y (BoehringerMannheim) to the tryptic digest adsorbed on the surface and incubate atroom temperature in a moist chamber for 5 min. Wash away the enzyme andsalt with water, add 1 ul of sinapinic acid and analyze by massspectrometry. FIG. 9A shows the composite mass spectra of human andbovine histidine-rich glycoprotein adsorbed on IDA-Cu(II) Sepharosebefore and after N-glycanase digestion. The mass shifts represent theremoval of carbohydrate from the respective glycoproteins. FIG. 9B showsthe composite mass spectra of trypsin digested peptides from thedeglycosylated proteins of the two species (top profile for humanprotein, second from bottom profile for bovine protein) and in situCu(II)-binding of the trypsin digested peptides of the two species(second from top profile for human protein, bottom profile for bovineprotein; the numbers 1, 2 indicate the number of copper bound). FIG. 9Cshows that one such Cu(II)-binding peptide (bottom profile) has at least4 His residues which are specifically modified by diethylpyrocarbonateto form 4 N-carbethoxy-histidyl adducts (1-4, top profile). FIG. 9Dshows the partial C-terminal sequence of the major Cu-binding peptide inthe bovine histidine rich glycoprotein. This example illustrates theeffective use of SEAC to probe the structure and function ofmetal-binding domains of proteins from different species.

II. Surface immobilized antibody as the affinity capture device

1. Polyclonal rabbit anti-human lactoferrin antibody is custom generatedagainst purified human lactoferrin by Bethyl Laboratories (Montgomery,Tex.). The antibody is affinity-purified by thiophilic adsorption andimmobilized lactoferrin columns. Sheep anti-rabbit IgG covalentlyattached to magnetic beads are obtained from Dynal AS, Oslo, Norway(uniform 2.8 μm supermagnetic polystyrene beads, ligand density 10 μgsheep IgG per mg bead). Human lactoferrin (1 nmole, ⁵⁹ Fe-labeled,81,100 Da) is incubated with rabbit anti-human lactoferrin antibody in20 mM sodium phosphate, 0.15M sodium chloride, pH 7.0 at 37° C. for 30min. Subsequently, 40 μl of sheep anti-rabbit IgG on Dynabeads (6-7×10⁸beads/ml) is added and incubated at 37° C. for 30 min. The beads arewashed with 500 μl of sodium phosphate buffer three times and 500 μlwater two times. The final amount of human lactoferrin bound to thecomplex is estimated to be 4 pmole. Approximately one-tenth of the beadsis transferred to a teflon-coated magnetic probe tip, mixed with 2 μl ofsinapinic acid (dissolved in 30% methanol, 0.1% trifluoroacetic acid)and analyzed by laser desorption time-of-flight mass spectrometry. FIG.10 shows the presence of lactoferrin (81,143 Da) in the antigen-primaryantibody-secondary antibody complex (upper profile), whereas the primaryantibody-secondary antibody control (lower profile) shows only therabbit antibody signal (149,000 Da for singly charged, and 74,500 Da forthe doubly charged).

This example illustrates that a) laser desorption is successfullycarried out on analyte affinity-adsorbed on surface immobilized antibody(if the analyte signal is unambiguously identified in a mixture ofprimary antibody-analyte complex, any capture device, e.g., surfaceimmobilized secondary antibody, Protein A, Protein G, Streptavidin, ofthe primary antibodies is used in this method of identifying theanalyte); b) the principle of protein discovery via specific molecularrecognition events where one of the analytes is detected through itsassociation with the primary target of capture; and c) the use ofmagnetic surface as efficient capture device.

2. Affinity-purified rabbit anti-human lactoferrin is covalently boundto the tip of an activated nylon probe element (2 mm diameter) viaglutaraldehyde. This is immersed in 1 ml of preterm infant urine, pH7.0, containing 350 fmole of human lactoferrin and stirred at 4-8° C.for 15 hr. The nylon probe tip is removed and washed with 1 ml of 20 mMsodium phosphate, 0.5M sodium chloride, 3M urea, pH 7.0 three times and1 ml of water two times. An aliquot of 2 μl of sinapinic acid (dissolvedin 30% methanol, 0.1% trifluoroacetic acid) is added and the sample isanalyzed by laser desorption time-of-flight mass spectrometry. FIG. 11Ashows the human lactoferrin molecular ion (signal/noise=2.5, average of25 laser shots) in the mass spectrum. FIG. 11B shows the equivalent massspectrum of whole preterm infant urine containing 1 nmole/ml oflactoferrin; the signal suppression caused by the presence of othercomponents in the urine sample is so severe that even addition ofseveral thousand fold excess over 350 fmole/ml of lactoferrin asdescribed for FIG. 11A can not be detected.

This example illustrates the use of a SEAC device on a flat surface (atwo-dimensional configuration) of a flexible probe element. This SEACdevice may be used to isolate target analyte materials fromundifferentiated biological samples such as blood, tears, urine, saliva,gastrointestinal fluids, spinal fluid, amniotic fluid, bone marrow,bacteria, viruses, cells in culture, biopsy tissue, plant tissue orfluids, insect tissue or fluids, etc. The specific affinity adsorptionstep cleaned up the analyte from contamination by other components in acomplex sample and thus overcome the signal depression effect especiallywhen the analyte is present in very low concentration (femtomole/ml).

3. Further improvement of detection sensitivity by the SEAC technique isachieved by amplification of a label bound to the analyte. One way ofdoing this is by the combination of enzyme catalysis and thestreptavidin-biotin system. After capturing minute quantities oflactoferrin on a nylon probe element as described in Example 3.II.2.biotinylated anti-lactoferrin antibody or biotinylated single-strandedDNA is used to bind specifically to the lactoferrin. Streptavidin isthen added to bind specifically to the biotinylated label. Finallybiotinylated alkaline phosphatase is added to bind specifically to thestreptavidin. Since several such biotinylated alkaline phosphatase canbind to one streptavidin, there is a primary level of amplification. Thesecond level of amplification comes from the enzyme catalysis where theenzyme can achieve a turnover number of 10² to 10³ min⁻¹. Assay ofalkaline phosphatase enzyme activity can easily be accomplished by usinga low molecular weight phosphorylated substrate such as ATP, NADPH or aphosphopeptide. The efficiency of detecting the mass shift of a lowmolecular weight analyte is much higher than that of a 80 kDaglycoprotein.

4. The ultimate improvement of detection at the present moment isachieved by the amplification based on the polymerase chain reactionprinciple. After capturing minute quantities of lactoferrin on a nylonprobe element as described in Example 3.II.2. biotinylatedanti-lactoferrin antibody or biotinylated single-stranded DNA is used tobind specifically to the lactoferrin. Streptavidin is then added to bindspecifically to the biotinylated label. A piece of biotinylated linearDNA is finally added to bind to the streptavidin. This bound DNA labelis amplified in a 30-cycle polymerase chain reaction procedure. Eachcycle consists of a 1 min denaturation step at 94° C., a 1 min annealingreaction at 58° C., and a 1 min primer extension reaction at 72° C. Thistechnique provides amplification factors in the 10⁶ fold range. Theamplified DNA is detected directly by laser desorption mass spectrometryusing 3-OH picolinic acid as the matrix.

5. Polyclonal rabbit anti-bovine histidine rich glycoprotein antibody iscustom generated against purified bovine histidine rich glycoprotein byBethyl Laboratories (Montgomery, Tex.). The antibody isaffinity-purified by thiophilic adsorption and immobilized bovinehistidine rich glycoprotein columns. The purified antibody isimmobilized on AffiGel 10 (BioRad Laboratories, Hercules, Calif., liganddensity 15 μmole/ml gel) according to manufacturer's instruction. Analiquot of 200 μl of bovine colostrum is diluted with 200 μl of 20 mMsodium phosphate, pH 7.0 and mixed with 50 μl of immobilized antibody at23° C. for 30 min. The gel is washed with 500 μl of 20 mM sodiumphosphate, 0.5M sodium chloride, 3M urea, pH 7.0 three times and 500 μlof water two times. An aliquot of 1 μl of the washed gel is mixed with 2μl of sinapinic acid (dissolved in 30% methanol, 0.1% trifluoroaceticacid) on the probe tip and analyzed by laser desorption time-of-flightmass spectrometry. FIG. 12 shows the composite mass spectra of purifiedbovine histidine rich glycoprotein (lower profile) and proteins affinityadsorbed from bovine colostrum (upper profile). The result indicates thepresence of intact histidine rich glycoprotein and its major proteolyticfragments in bovine colostrum.

This example illustrates the effective use of SEAC as a fast and simpletechnique to detect and characterize new proteins in a small quantity ofbiological fluid. This result supports the initial findings obtained bythe very labor-intensive technique of immunoblotting of polyacrylamidegel electrophoresis.

6. Antibody epitope mapping is easily achieved with the SEAC technique.Three different sources of anti-human follicle stimulating hormone (apolyclonal specific against beta FSH from Chemicon International,Temecula, Calif., a monoclonal specific against beta 3 epitope fromSerotec, Indianapolis, Ind., a monoclonal from Biodesign, Kennebunk,Me.) are immobilized on AffiGel 10 according to manufacturer'sinstruction. These immobilized antibodies are all tested to bindspecifically the follicle stimulating hormone by incubating with twodifferent preparations of follicle stimulating hormone (a semipurepreparation from Chemicon, and a crude preparation from AccurateChemical and Scientific Corp.) and then analyzed by mass spectrometry inthe presence of sinapinic acid. Then the semipure preparation of humanFSH (Chemicon) is digested with trypsin and separate aliquots (7 ul) arereacted with the immobilized antibodies (10 ul of 1:1 gel suspension) inphosphate-buffered saline at 4° C. for 2 hr. After washing withphosphate-buffered saline and water, the adsorbed proteins are analyzedby laser desorption mass spectrometry in the presence of sinapinic acid.FIG. 13 shows the composite mass spectra of the peptides of folliclestimulating hormone recognized by the different antibodies. The twomonoclonal antibodies clearly recognize different epitopes, whereas thepolyclonal recognizes multiple epitopes common to those recognized byboth monoclonals.

III. Surface Immobilized Nucleic Acid as the Affinity Capture Device

1. Single-stranded DNA immobilized on 4% agarose beads are obtained fromGIBCO BRL (Gaithersburg, Md., ligand density 05-1.0 mg DNA/ml gel). Analiquot of ¹²⁵ I-human lactoferrin (equivalent to 49 nmole) is mixedwith 100 μl of immobilized single-stranded DNA in 20 mM HEPES, pH 7.0 atroom temperature for 10 min. The gel is washed with 500 μl of HEPESbuffer five times and then suspended in equal volume of water. Theamount of lactoferrin bound per bead is estimated to be 62 fmole bydetermining the radioactivity and counting the number of beads per unitvolume. Various numbers of beads (from 1 to 12) are deposited on 0.5 mmdiameter probe tips, mixed with 0.2 μl of sinapinic acid (dissolved in30% methanol, 0.1% trifluoroacetic acid) and analyzed by laserdesorption time-of-flight mass spectrometry. FIG. 14 shows the massspectrum of lactoferrin affinity adsorbed on a single bead ofsingle-stranded DNA agarose. This is a representative spectrum from atotal of five (average of 100 laser shots per spectrum) obtained fromthe single bead.

This example illustrates that laser desorption is successfully carriedout on analyte affinity adsorbed on surface immobilized biopolymer suchas nucleic acid. The specificity of interaction between humanlactoferrin and DNA has been documented and effectively exploited incapturing minute quantities of lactoferrin from preterm infant urine. Inthis case, the combination of the efficiency of transferring thelactoferrin affinity capture device with the sensitivity of laserdesorption mass spectrometry greatly increases the sensitivity ofdetection.

2. An aliquot of 1 ml of preterm infant urine containing 30 pmole of ⁵⁹Fe-human lactoferrin is mixed with 20 μl of single-stranded DNA agarosein 0.1 M HEPES pH 7.4 at 23° C. for 15 min. The gel is washed with 500μl of HEPES buffer two times and 500 μl of water two times. The gel issuspended in equal volume of water and 1 μl of the suspension(containing not more than 350 fmole of adsorbed lactoferrin asdetermined by radioactivity) is mixed with 1 μl of sinapinic acid(dissolved in 30% methanol, 0.1% trifluoroacetic acid) on a probe tipand analyzed by laser desorption time-of-flight mass spectrometry. FIG.15 shows the mass spectrum of lactoferrin extracted from urine bysurface immobilized DNA as the affinity capture device.

This example illustrates the efficiency and sensitivity of detectingminute quantities of high molecular weight analyte in biological fluidwith the DNA capture device.

IV. Surface immobilized miscellaneous biomolecule as the affinitycapture device

1. Soybean trypsin inhibitor (Sigma) is immobilized on AffiGel 10(BioRad) according to manufacturer's instructions. An aliquot of 100 μlof human duodenal aspirate is mixed with 50 μl of surface immobilizedsoybean trypsin inhibitor at pH 7.0 (20 mM sodium phosphate, 0.5M sodiumchloride) at 23° C. for 15 min. The gel is then washed with 500 μl ofphosphate buffer three times and 500 μl of water two times. Aliquots of1 μl of gel suspension or the original duodenal aspirate are mixed with2 μl of sinapinic acid (dissolved in 50% acetonitrile, 0.1%trifluoroacetic acid) and analyzed by laser desorption time-of-flightmass spectrometry. FIG. 16A shows the composite mass spectra of thetotal duodenal aspirate (lower profile) and the proteins adsorbed bysurface immobilized soybean trypsin inhibitor (upper profile). The majorpeak in the affinity captured sample represents trypsin. Similar resultsare obtained with only 1 μl of duodenal fluid deposited on a) the tip ofa nylon probe element coupled to soybean trypsin inhibitor viaglutaraldehyde and b) the tip of an acrylic probe element coated withpolyacrylamide coupled to soybean trypsin inhibitor via eitherglutaraldehyde or divinyl sulfone (FIG. 16B).

These results indicate a) the unambiguity in detecting andcharacterizing a specific analyte in biological fluids and b) thefeasibility of in situ sampling by inserting a flexible (e.g. nylon)probe element through an endoscope directly into the human body (e.g.small intestine) for diagnostic purposes.

2. Streptavidin immobilized on Dynabead (uniform, 2.8 μm,superparamagnetic, polystyrene beads) is obtained Dynal, AS, Oslo,Norway. Aliquots of 150 μl of human plasma or urine containing 18 pmoleof biotinylated insulin (Sigma) are mixed with 20 μl suspension ofstreptavidin Dynabead at pH 7.0 (20 mM sodium phosphate, 0.5M sodiumchloride) at 23° C. for 10 min. The beads are then washed with 500 μlbuffer containing 3M urea three times and 500 μl water once. Aliquots of0.5 μl of the bead suspension are mixed with 2 μl of sinapinic acid(dissolved in 30% methanol, 0.1% trifluoroacetic acid) and analyzed bylaser desorption time-of-flight mass spectrometry. FIG. 17A shows themass spectrum of biotinylated insulin affinity adsorbed from urine. Themultiple peaks represent insulin derivatized with one to three biotingroups. FIG. 17B shows the mass spectrum of biotinylated insulinaffinity adsorbed from plasma.

This example illustrates that laser desorption is carried out on analyteaffinity adsorbed via the biotin-streptavidin binding. In view of thetight binding between biotin and avidin (Ka=10¹⁵ M⁻¹), this systemserves as an ideal SEAC device for proteins and nucleic acid on a probesurface where in situ sequential chemical and enzymatic modificationsare performed.

3. Human estrogen receptor DNA-binding domain (84 residues) is expressedin bacteria. The plasmid expression vector pT₇ ERDBD (J. Schwabe, MRCLaboratory of Molecular Biology, Cambridge, UK) is transformed into E.coli BL21(DE3)plyS cells (Novagene). Expression of the DNA bindingdomain is induced by 1 mM isopropylthiogalactoside (GIBCO BRL) and thebacteria are harvested after induction for 3 hr. Whole induced bacteriaare analyzed directly by matrix-assisted laser desorption/ionizationmass spectrometry to verify that the DNA-binding domain is the majorpeptide synthesized. The peptide is purified by reverse phase HPLC fromthe bacterial lyzate, and immobilized on AffiGel 10 (BioRad). A 30-bpDNA sequence containing the estrogen response element is synthesized byGenosys (Houston, Tex.). Interaction of surface affinity adsorbed apo-,Zn- and Cu-bound forms of DNA-binding domain with sequence specificnucleic acid (estrogen response element) are studied on glass probeelements using 3-hydroxypicolinic acid as the matrix.

This example illustrates the use of protein surface functional domain ascapture device in SEAC. The effect of metal-binding on the structure andfunction of such protein surface domains can be investigated.

4. Different aliquots of lectins immobilized on surfaces (e.g., ConA-Sepharose, wheat germ lectin-Sepharose, Pharmacia) are used to bindthe glycopeptides in human and bovine histidine-rich glycoproteintryptic digests. After washing with buffers and water to remove unboundpeptides, sequential enzyme digestion are performed in situ with FUCaseI, MANase I, HEXase I, NANase III and PNGase (Glyko, Inc, Novato,Calif.). The samples are analyzed with laser desorption time-of-flightmass spectrometry to study the carbohydrate composition of theglycopeptides in the two proteins. This example illustrates the use ofSEAC device to tether a glycopeptide, the carbohydrate component ofwhich can then be sequenced in situ.

V. Surface Immobilized Dye as the Affinity Capture Device

Cibacron Blue 3GA-agarose (Type 3000, 4% beaded agarose, ligand density2-5 pmoles/ml gel) is obtained from Sigma. An aliquot of 200 μl of humanplasma is mixed with 50 μl of surface immobilized Cibacron Blue at pH7.0 (20 mM sodium phosphate, 0.5M sodum chloride) at 23° C. for 10 min.The gel is then washed with 500 μl of buffer three times and 500 μl ofwater two times. An aliquot of 1 μl of gel suspension is mixed with 2 μlof sinapinic acid (dissolved in 50% acetonitrile, 0.1% trifluoroaceticacid) and analyzed by laser desorption time-of-flight mass spectrometry.FIG. 18 shows the selective adsorption of human serum albumin (doublycharged ion [M+2H]²⁺, 32,000 m/z, singly charged ion [M+H]⁺, 64,000 m/z,dimer ion, 2[M+H]⁺, 128,000 m/z) from the serum sample by surfaceimmobilized Cibacron Blue (lower profile). Other immobilized dyes testedincluded Reactive Red 120-agarose, Reactive Blue-agarose, ReactiveGreen-agarose, Reactive Yellow-agarose (all from Sigma) and each selectsdifferent proteins from human plasma.

EXAMPLE 4 Surface Enhanced Neat Desorption (SEND)

This example describes the method for desorption and ionization ofanalytes in which the analyte is not dispersed in a matrix crystallinestructure but is presented within, on or above an attached surface ofenergy absorbing molecules in a position where it is accessible andamenable to a wide variety of chemical, physical and biologicalmodification or recognition reactions. The surface is derivatized withthe appropriate density of energy absorbing molecules bonded (covalentlyor noncovalently) in a variety of geometries such that mono layers andmultiple layers of attached energy absorbing molecules is used tofacilitate the desorption of analyte molecules of varying masses.

The Examples shown below (Groups I-IV) demonstrate the combined SEND andSEAC where the adsorbed (bonded) energy absorbing molecules also act asaffinity adsorption reagents to enhance the capture of analytemolecules.

I. Energy Absorbing Molecules Bound by Covalent Bond to the Surface

1. Cinnamamide (Aldrich) (not a matrix at laser wavelength of 355 nm byprior art) is dissolved in isopropanol: 0.5M sodium carbonate (3:1) andmixed with divinyl sulfone (Fluka, Ronkonkoma, N.Y.) activated Sepharose(Pharmacia) at 23° C. for 2 hr. The excess energy absorbing moleculesare washed away with isopropanol. The proposed molecular structure ispresented in FIG. 19. Aliquots of 2 μl of the bound or free moleculesare deposited on the probe tips, 1 μl of human estrogen receptordimerization domain in 0.1% trifluoroacetic acid is added on top andanalyzed by laser desorption time-of-flight mass spectrometry. Theresult shows that peptide ion signals are detected only on the boundenergy absorbing molecule surface (FIG. 20, top profile), the freemolecules are not effective (FIG. 20, bottom profile).

2. Cinnamyl bromide (Aldrich) (not a matrix at laser wavelength of 355nm by prior art) is dissolved in isopropanol:0.5M sodium carbonate (3:1)and mixed with divinyl sulfone (Fluka) activated Sepharose at 23° C. for15 hr. The excess energy absorbing molecules are washed away withisopropanol. The proposed molecular structure is presented in FIG. 21.Aliquots of 2 μl of the bound or free molecules are deposited on theprobe tips, 1 μl of peptide mixtures in 0.1% trifluoroacetic acid isadded on top and analyzed by laser desorption time-of-flight massspectrometry. The result shows that peptide ion signals are detectedonly on the bound energy absorbing molecule surface (FIG. 22, topprofile), the free molecules are not effective (FIG. 22 bottom profile).

3. Dihydroxybenzoic acid is activated by dicyclohexylcarbodiimide andmixed with Fmoc-MAP 8 branch resin (Applied Biosystems, Forster City,Calif.) at 23° C. for 15 hr. The excess energy absorbing molecules arewashed away by methanol. The proposed molecular structure is presentedin FIG. 23. Aliquots of 1 μl of the MAP 8 branch surface with andwithout bound energy absorbing molecules are deposited on the probetips, 1 μl of peptide mixtures in 0.1% trifluoroacetic acid was added ontop and analyzed by laser desorption time-of-flight mass spectrometry.The result shows that peptide ion signals are detected only on thesurface with bound energy absorbing molecules (FIG. 24, bottom profile),the control surface without any energy absorbing molecules is noteffective (FIG. 24, top profile).

4. α-cyano-4-hydorxycinnamic acid is dissolved in methanol and mixedwith AffiGel 10 or AffiGel 15 (BioRad) at various pHs at 23° C. for 2-24hours. The excess energy absorbing molecules are washed away bymethanol. Aliquots of 2 μl of the bound molecules are deposited on theprobe tips, 1 μl of peptide mixtures or myoglobin, or trypsin orcarbonic anhydrase is added on top and analyzed by laser desorptiontime-of-flight mass spectrometry. The result shows that myoglobin ionsignal is detected on the surface with bound energy absorbing molecules(FIG. 25) with very little contaminating low mass ion signals (FIG. 25).

5. A 40% polyacrylamide solution is prepared and cast into the desiredshape of a probe tip. The gel is allowed to air dry until no noticeablereduction in size is observed. The tip is submerged into a 9%glutaraldehyde/buffer (v/v) solution and incubated with gentle shakingat 37° C. for 2 hours. After incubation, buffer is used to rinse offexcess glutaraldehyde. The activated tip is added to a saturatedbuffered energy absorbing molecule solution and incubated at 37° C.(approx.) for 24 hours (approx.) with gentle shaking. Organic solventsare used to solubilize the energy absorbing molecules in situations thatrequired it. The tip is rinsed with buffer and placed into a 9%ethanolamine/water (v/v) solution to incubate at 25° C. with gentleshaking for 30 minutes. Next, the tip is rinsed with buffer and added toa 5 mg/mL solution of sodium cyanoborohydride/buffer to incubate at 25°C. for 30 minutes. Finally, the tip is rinsed well with buffer andstored until use. The same reaction is carried out on nylon tips whichis prepared by hydrolysis with 6N HCl under sonication for 2 minutes andthen rinsed well with water and buffer. The same reaction is alsoperformed on acrylic tips activated by soaking in 20% NaOH for 7 dayswith sonication each day for 30-60 min and then washed. The proposedgeneral molecular structure of the surface is shown in FIG. 26.

6. A 40% polyacrylamide solution is prepared and cast into the desiredshape of a probe tip. The gel is air dried until no noticeable reductionin size is observed. A 0.5M sodium carbonate buffer with a pH of 8.8 isprepared as rinsing buffer. The tip is next placed into a solution ofdivinyl sulfone (Fluka) and buffer at a ratio of 10:1, respectively andincubated for 24 hours. The tip is rinsed with buffer and placed into anenergy absorbing molecule buffered solution at a pH of 8 to incubate for2 hours. The same reaction is carried out on nylon tips which isprepared by hydrolysis with 6N HCl under sonication for 2 minutes andthen rinsed well with water and buffer. The same reaction is alsoperformed on acrylic tips activated by soaking in 20% NaOH for 7 dayswith sonication each day for 30-60 min and then washed. The proposedgeneral molecular structure of the surface is shown in FIG. 27.

7. A 40% polyacrylamide solution is prepared and cast into the desiredshape of a probe tip. The gel is air dried until no noticeable reductionin size is observed. An energy absorbing molecule solution at 100 mg/mLin dichloromethane/NMP (2:1 respectively) and a 1Mdicyclohexylcarbodiimide/NMP solution are mixed at a ratio of 1:2(EAM:DCC), respectively. The EAM/DCC solution is next incubated at 25°C. for 1 hour while stirring. After incubation, a white precipitate isobserved. The white precipitate is filtered in a sintered glass filter.The flow through is the DCC activated EAM. Next, the tip is placed intothe DCC activated EAM solution and incubated at 25° C. for 2 hours(approx.). The tip is finally rinsed with a variety of solvents such asacetone, dichloromethane, methanol, NMP, and hexane. The same reactionis carried out on nylon tips which is prepared by hydrolysis with 6N HClunder sonication for 2 minutes and then rinsed well with water andbuffer. The same reaction is also performed on acrylic tips activated bysoaking in 20% NaOH for 7 days with sonication each day for 30-60 minand then washed. The proposed general molecular structure of the surfaceis shown in FIG. 28.

8. A 40% polyacrylamide solution is prepared and cast into the desiredshape of a probe tip. The gel is air dried until no noticeable reductionin size was observed. A 100 mg/mL solution of N-α-Fmoc-N-ε-Fmoc-L-lysinein dichloromethane/NMP (2:1 respectively) and a 1M DCC/NMP solution aremixed at a ratio of 1:2 (lysine:DCC), respectively. The lysine/DCCsolution is incubated at 25° C. for 1 hour while stirring. Afterincubation, a white precipitate is observed and filtered with a sinteredglass filter. The flow through is DCC activated lysine. The tip isplaced into the DCC activated lysine solution and incubated at 25° C.for 2 hours (approx.). The tip is next placed into 5 mL of piperidineand incubated at 25° C. for 45 minutes with gentle stirring. DCCactivated lysine is repeatedly reacted in consecutive cycles with thetip until the desired lysine branching is attained. An EAM solution at100 mg/mL in dichloromethane/NMP (2:1 respectively) and a 1M DCC/NMPsolution are mixed at a ratio of 1:2 (EAM:DCC), respectively. TheEAM/DCC solution is incubated at 25° C. for 1 hour while stirring. Afterincubation, a white precipitate is observed and filtered with a sinteredglass filter. The flow through is the DCC activated EAM. The EAMcontains an acid functional group that reacts with the DCC. The tip isplaced into the DCC activated EAM solution and incubated at 25° C. for 2hours (approx.) with gentle shaking. Finally, the tip is rinsed withexcess dichloromethane, NMP, and methanol before use. The same reactionis carried out on nylon tips which is prepared by hydrolysis with 6N HClunder sonication for 2 minutes and then rinsed well with water andbuffer. The same reaction is also performed on acrylic tips activated bysoaking in 20% NaOH for 7 days with sonication each day for 30-60 minand then washed. The proposed general molecular structure of the surfaceis shown in FIG. 29.

II. Energy Absorbing Molecules Bound by Co-ordinate Covalent Bond to theSurface

1. Thiosalicylic acid (Aldrich) is dissolved in either water or 50%methanol in water or methanol. These solutions are either used as suchor the pH of the solutions is adjusted to 6.5 with 0.5M sodiumbicarbonate or ammonium hydroxide or triethylamine. Cu(II) ion arechelated by either iminodiacetate (IDA) (Chelating Sepharose Fast Flow,Pharmacia) or tris(carboxymethyl)ethyleneidamine (TED) (synthesized asdescribed by Yip and Hutchens, 1991) immobilized on gel surface. Thesolutions of energy absorbing molecule are mixed with the IDA-Cu(II) orTED-Cu(II) gel at 4° to 23° C. for 5 min to 15 hours. The excess energyabsorbing molecules are washed away with either water or 50% methanol inwater or methanol. The proposed molecular structure of the surface isshown in FIG. 30. Aliquots of 1 μl of the bound energy absorbingmolecules are deposited on the probe tips, 1 μl of peptide mixtures orestrogen receptor dimerization domain or myoglobin in 0.1%trifluoroacetic acid is added on top and analyzed by laser desorptiontime-of-flight mass spectrometry. FIG. 31 shows one representative massspectrum of estrogen receptor dimerization domain desorbed from thissurface.

2. Sequential in situ reactions are readily accomplished on samplesdeposited on top of an EAM surface. Thiosalicylic acid co-ordinatecovalently bound to IDA-Cu(II) on a probe surface is prepared asdescribed in Section 2.1. An aliquot of 1 μl of (GHHPH)₅ G peptide isdeposited on the surface and analyzed by laser desorption time-of-flightmass spectrometry. After obtaining several spectra (each an average of50 laser shots), the sample is removed. An aliquot of 2 μl ofcarboxypeptidase Y (Boehringer Mannheim) is added directly on thesurface and incubated at 37° C. in a moist chamber for 5 min to 1 hr.The in situ enzyme digestion is terminated by 1 μl of 0.1%trifluoroacetic acid and the sample is reanalyzed by mass spectrometry.

3. Another illustration of sequential in situ reaction is trypsindigestion followed by C-terminal sequencing. Thiosalicylic acidco-ordinate covalently bound to IDA-Cu(II) on a probe surface isprepared as described in Section 2.1. An aliquot of 1 μl of estrogenreceptor dimerization domain (6168.4 Da) is deposited on the surface andanalyzed by laser desorption time-of-flight mass spectrometry. Afterobtaining several spectra (each an average of 20 laser shots), thesample is removed. An aliquot of 2 μl of trypsin (Sigma) in 0.1M sodiumbicarbonate is added on the surface and incubated at 37° C. for 15 min.The in situ enzyme digestion is terminated by 1 μl of 0.1%trifluoroacetic acid and the sample is reanalyzed by mass spectrometry.After obtaining several spectra (each an average of 20 laser shots), thesample is removed. An aliquot of 2 μl of carboxypeptidase Y (BoehringerMannheim) is added directly on the surface and incubated at 37° C. in amoist chamber for 1 hr. The in situ enzyme digestion is terminated by 1μl of 0.1% trifluoroacetic acid and the sample is reanalyzed by massspectrometry.

III. Energy Absorbing Molecules Bound by Ionic Bond to the Surface

Sinnapinic acid or α-cyano-4-hydroxycinnamic acid are suspended in waterand the pH is adjusted to 6.6 with dilute sodium hydroxide. TentacleDEAE Fractogel (EM Separations, Gibbstown, N.J.) is washed with 20 mMHEPES, pH 6.0 and suction dried. The energy absorbing molecules solutionis mixed with the DEAE gel at 23° C. for 15 hours. The gel is washedwith water until all excess energy absorbing molecules were removed. Theproposed molecular structure of the surface is shown in FIG. 32. Analiquot of 0.5 μl of the bound energy absorbing molecules is depositedon the probe tips, 1 μl of estrogen receptor dimerization domain ormyoglobin in 0.1% trifluoroacetic acid is added on top and analyzed bylaser desorption time-of-flight mass spectrometry. FIGS. 33A and B showthe mass spectra.

IV. Energy Absorbing Molecules Bound by Hydrophobic/Van der Waals Bondsto the Surfaces

1. α-cyano-4-hydroxcinnamic acid is dissolved in 50% methanol in waterand dimethylsulfoxide. This is mixed with aminomethylated polystyrene at23° C. for 15 hours. The excess energy absorbing molecules are washedaway with 50% methanol in water. The proposed molecular structure isshown in FIG. 34. An aliquot of 1 μl of the bound energy absorbingmolecules is deposited on the probe tip, 1 μl of peptide is added on topand analyzed by laser desorption time-of-flight mass spectrometry.

EXAMPLE 5 Surfaces Enhanced for Photolabile Attachment and Release(SEPAR)

The linear assembly of individual building blocks (monomers) that definethe structure and characteristics of biopolymers such as DNA, RNA, andprotein are often unknown but are decoded or sequenced (in whole or inpart) with a method that involves differential mass determinations ofpartially digested (i.e., chemical or enzymatic) biopolymer analytes bylaser desorption/ionization time-of-flight (TOF) mass spectrometry (MS).

Given biopolymers are first coupled to the SELDI probe element surfacethrough one or more (multiple) covalent photolytic (i.e., lightsensitive) bonds. Next, various number of individual units (monomers) atthe ends of the biopolymer are cleaved (i.e., removed) in a singlereaction by enzymatic or chemical methods. The analytes remaining on theprobe element surface consist of a variety (population) of mass-definedbiopolymers with different numbers of their end monomer units missing. Asmall but sufficient portion of the modified biopolymers are uncoupled(untethered) from the probe element surface by laser light, that is, bycleavage of the photolytic bonds with UV light between 260 nm and 365nm. The uncoupled biopolymers are desorbed/ionized by time-of-flightmass spectrometry.

I. Coupling of Biopolymers to the SELDI Surface

Three components are involved: 1) a surface that is activated to reactwith either amine or carboxyl groups, or both; 2) photolytic compound,typically azo-based compound of the general formula R₁ --N═N--R₂, e.g.,5-(4-aminophenylazo)salicylic acid (Aldrich), azodicarbonamide(Aldrich), or other mechanisms generating such photolytic bond such asthe active hydrogen reactive chemistries with diazonium compounds areused; and 3) biopolymer, e.g., proteins, nucleic acids, carbohydrates.

A photolytic compound must first be attached to activated surface, e.g.,azodicarbonamide to amine-reactive surfaces, aminophenylazosalicylicacid to either amine or carboxyl reactive surfaces. Then activate eitherphotolytic compound or biopolymer by one of many conventionalchemistries, e.g., amine reactive chemistries--cyanogen bromide,N-hydroxysuccinimide esters, FMP activation, EDC-mediated, divinylsulfone; hydroxyl reactive chemistries--epoxy activation, divinylsulfone; sulfhydryl reactive chemistries--iodoacetyl activation,maleimide, divinyl sulfone, epoxy activation; carbonyl reactivechemistries--hydrazide, reductive amination; active hydrogen reactivechemistries--diazonium, which also generate a photolytic azo bond at thesame time. Finally, attach the biopolymer to photolytic compound throughone or more (multiple) bonds. Wash away the excess chemicals withaqueous and organic solvents, high ionic strength and low pH solvents inmultiple cycles.

II. Mass Spectrometric Analysis to Verify Structural Integrity

UV laser from 260 to 365 nm will cleave the photolytic bond. Theuncoupled biopolymers are desorbed/ionized by MALDI TOF (one skilled inthe art knows that SEND, SEAC and SEPAR may also be used).

III. In situ Sequencing of Biopolymer

This is accomplished by any of the well-known sequential degradationwith enzymatic or chemical methods, e.g., N-terminal sequencing ofproteins with aminopeptidase, C-terminal sequencing of proteins withcarboxypeptidase, N-terminal sequencing of proteins with Edmandegradation; sequencing of nucleic acids with exonuclease, sequencing ofnucleic acids with Sanger's method; sequencing of carbohydrate withspecific enzymes such as neuraminidase, mannase, fucase, galactosidase,glucosidase, O- or N-glycanase. After washing to remove excess reagentand reaction products, the analytes remaining tethered on the surfacevia multiple photolytic bonds consisting of a population of mass-definedbiopolymers with different numbers of their end monomer missing areanalyzed by MALDI TOF MS (one skilled in the art knows that SEND, SEACand SEPAR may also be used).

Multiple internal sequencing with enzymatic or chemical methods, e.g.,cleavage of proteins with endoprotease or cyanogen bromide followed bysequential degradation of N- and/or C-terminal; cleavage of nucleicacids with endonuclease followed by sequential degradation withexonuclease or chemical method; cleavage of polysaccharide chains withendoglycosidase H or endoglycosidase F followed by sequential cleavagewith specific enzymes. After washing to remove excess reagent andreaction products, the analytes remaining on the surface consisting ofmultiple populations of mass-defined biopolymers with different numbersof their end monomer missing are analyzed by MALDI TOF MS (one skilledin the art knows).

IV. Specific Examples of Sequencing

A demonstration of this principle is provided by the actual amino acidsequence determination of a 26-residue peptide:

GHHPHGHHPHGHHPHGHHPHGHHPHGHHPHG.

This peptide (GHHPH)₅ G defines the metal-binding domain within theintact sequence of the 80-kDa protein known as histidine-richglycoprotein (HRG).

Glass beads with surface arylamine groups as coupling ligands (Sigma)are washed with and suspended in cold 0.3M HCl. A 50 mg/mL aqueoussolution of NaNO₂ is added to the beads at a ratio of 1:5 (v/v) (NaNO₂:HCl) and incubated at 4° C. for 15 minutes with gentle shaking. Afterincubation, the beads are washed with cold 0.3M HCl and 50 mM sodiumphosphate buffer pH 8.0. The peptide to be sequenced is added to thebeads in sodium phosphate buffer at pH 8.0 and incubated for 24 hrs. at4° C. with gentle shaking. The beads with coupled peptides are washedwith sodium phosphate buffer, sodium phosphate buffer with highconcentration of salt (e.g., 1.0M), dilute acid and organic solvent(e.g., methanol) until no peptide signal is detected in the supernate byMALDI-TOF mass spectrometry (one skilled in the art knows SEND, SEAC,and SEPAR may also be used) or by absorbance at 220 nm. An aliquot of 1μL of the beads is then deposited on the probe tip, 1 μL of sinapinicacid (dissolved in 50% methanol/0.1% trifluoroacetic acid) is mixed withthe beads and the sample was analyzed by laser desorption time-of-flightmass spectrometry. After obtaining several spectra (each an average of50 laser shots), the remaining peptides on the surface are washed freeof sinapinic acid with methanol and then digested with carboxypeptidaseY (Boehringer Mannheim) at 23° C. in a moist chamber. The digestedpeptides are next washed with phosphate buffered saline (PBS) pH 8.0. Analiquot of 1 μL of sinapinic acid is added to the surface and analyzedagain by laser desorption time-of-flight mass spectrometry. The resultof the C-terminal sequence analysis of the GHHPHG sequence is shown inFIG. 35. A nascent sequence of the peptide is observed. The sequence isdeduced by the differences in the mass between two peaks.

The second example is the simultaneous sequencing of multiple peptidescovalently bound by photolytic bonds to a surface. Human estrogenreceptor dimerization domain (6168.4 Da) is tethered to the surface, viamultiple photolytic bonds. The peptide has three methionine residues inits sequence and are cleaved specifically by cyanogen bromide togenerate peptides of masses 2170.58 Da (D1-M18), 3118.77 Da (A19-M45),535.62 Da (S46-M50) and 397.62 Da (E51-L53). All these peptides arebound to the surface via the photolytic bonds. Each of these aresubsequently digested in situ with carboxypeptidase Y to generate anascent sequence that is completely resolved from the other.

Another approach to protein structure determination is simultaneousN-terminal sequencing of multiple peptides generated by tryptic digestof a protein coupled to a surface by multiple photolytic bonds. InsulinB chain is tethered to the surface via multiple photolytic bonds. Thepeptide has two lysinelarginine residues in its sequence that arecleaved specifically by trypsin to generate peptides of masses 2585.9 Da(F1-R22) and 859.0 Da (G23-K29), both of which are bound to the surfacevia the photolytic bonds. Each of these are subsequently subjected insitu to either aminopeptidase digestion or multiple cycles of Edmandegradation to generate a nascent sequence that is completely resolvedfrom the other.

Coupling and sequencing of nucleic acids is performed with similarprocedure. Glass beads with surface arylamine groups as coupling ligands(Sigma) are washed with and suspended in cold 0.3M HCl. A 50 mg/mLaqueous solution of NaNO₂ is added to the beads at a ratio of 1:5 (v/v)(NaNO₂ :HCl) and incubated at 4° C. for 15 minutes with gentle shaking.After incubation, the beads are washed with cold 0.3M HCl and 50 mMsodium phosphate buffer pH 8.0. The DNA (e.g., estrogen receptorresponsive element, a 30-base pair oligonucleotide) to be sequenced isadded to the beads in sodium phosphate buffer at pH 8.0 and incubatedfor 24 hrs. at 4° C. with gentle shaking. The beads with coupled DNA arewashed with sodium phosphate buffer, sodium phosphate buffer with highconcentration of salt (e.g., 1.0M), dilute acid and organic solvent(e.g., methanol) until no DNA signal is detected in the supernate byMALDI-TOF mass spectrometry (one skilled in the art knows that SEND,SEAC and SEPAR may also be used) or by absorbance at 260 nm. An aliquotof 1 μL of the beads is then deposited on the probe tip, 1 μL of3-hydroxypicolinic acid (dissolved in 50% methanol/0.1% trifluoroaceticacid) is mixed with the beads and the sample is analyzed by laserdesorption time-of-flight mass spectrometry. After obtaining severalmass spectra (each an average of 50 laser shots), the remaining DNAbound on the surface are washed free of 3-hydroxypicolinic acid withmethanol and digested with exonuclease (Boehringer Mannheim) at 23° C.in a moist chamber. The digested DNA on the surface are next washed withphosphate buffered saline (PBS) pH 8.0 to remove excess reagent andreaction products. An aliquot of 1 μL of 3-hydroxypicolinic acid isadded to the surface and analyzed again by laser desorptiontime-of-flight mass spectrometry. A nascent sequence of the DNA isgenerated. The sequence is deduced by the differences in the massbetween two peaks.

Carbohydrate chains are oxidized by periodate and activated to bespecifically coupled to a photolytic compound on a surface. Sequencingof the tethered carbohydrate with specific enzymes such asneuraminidase, mannase, fucase, galactosidase, glucosidase, O- orN-glycanase is carried out and determined by laser desorptiontime-of-flight mass spectrometry. 5-(4aminophenylazo)salicylic acid(Aldrich) is coupled to a carboxyl reactive surface such as arylamine oncontrolled pore glass beads. The carbohydrate moieties of human andbovine histidine rich glycoprotein are oxidized by low concentration(0.2M) of sodium meta-periodate in water at 23° C. for 90 min. Theexcess reagents are washed away with water. Add the proteins to the5-(4-aminophenylazo)salicylic acid coupled to controlled pore glassbeads in phosphate buffer, pH 8.0. Then add sodium cyanoborohydride (0.6mg(100 μl) and stir in a fume hood at 23° C. for 18 hr. Wash extensivelywith water, 1M NaCl, and then water again to remove excess reagents andunreacted proteins. An aliquot of 1 μL of the beads is then deposited onthe probe tip, 1 μL of sinapinic acid (dissolved in 50% methanol/0.1%trifluoroacetic acid) is mixed with the beads and the sample is analyzedby laser desorption time-of-flight mass spectrometry. The remainingproteins bound on the surface are washed free of sinapinic acid withmethanol and incubated with 2 μl of trypsin in phosphate buffer pH 8.0at 37° C. for 30 min. The surface with bound glycopeptides is washedthoroughly with phosphate buffered saline and water to remove excessreagent and unbound peptides. An aliquot of 1 μL of sinapinic acid ismixed with the beads and the sample is analyzed by laser desorptiontime-of-flight mass spectrometry. After obtaining several mass spectra(each an average of 50 laser shots), the remaining glycopeptides on theprobe surface are washed free of sinapinic acid with methanol anddigested in sequence or in combination with N-acetylneuraminidase(NANase III, Glyko, 50 mM sodium phosphate buffer, pH 6.0, 37° C. 1 hr),mannosidase (MANase I, Glyko, 50 mM sodium phosphate, pH 6.0, 37° C. 18hr), fucosidase (FUCase I, Glyko, 50 mM sodium phosphate, pH 5.0, 37° C.18 hr), N-acetylglucosaminidase (HEXase I, Glyko, 50 mM sodiumphosphate, pH 5.0, 37° C. 4 hr), O-glycosidase (Glyko, 50 mM sodiumphosphate, pH 5.0, 37° C. 18 hr) or N-glycanase (PNGase F, Glyko, 100 mMsodium phosphate, pH 8.6, 37° C., 18 hr). The fragmented glycopeptideson the surface are fmally washed with phosphate buffered saline andwater to remove the reagents and reaction products. An aliquot of 1 μLof sinapinic acid is added to the surface and analyzed again by laserdesorption time-of-flight mass spectrometry. Nascent sequences of theglycopeptides are observed. The sequences are deduced by the differencesin the mass between two peaks.

All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Theoligonucleotides, compounds, methods, procedures and techniquesdescribed herein are presently representative of the preferredembodiments, are intended to be exemplary and are not intended aslimitations on the scope. Changes therein and other uses will occur tothose skilled in the art which are encompassed within the spirit of theinvention and are defined by the scope of the appended claims.

What is claimed:
 1. A system for detecting an analyte comprising:aremovably insertable probe having a surface for presenting the analyteto an energy source that emits energy capable of desorbing the analytefrom the probe surface, and an immobilized affinity reagent on the probesurface capable of binding the analyte; an energy source that directsenergy to the probe surface for desorbing the analyte; and a detector incommunication with the probe surface that detects the desorbed analyte.2. The system of claim 1 which is a laser desorption mass spectometerwherein:the energy source emits laser light that ionizes the analyte toproduce an ion, the system further comprises means for accelerating theion to the detector, the detector detects the ion, and the systemfurther comprises means for determining the mass of the ion.
 3. Thesystem of claim 1 wherein the energy source emits laser light.
 4. Thesystem of claim 1 wherein the energy source emits plasma energy or fastatoms.
 5. The system of claim 1 wherein the energy source emits energyof a variety of wavelengths.
 6. The system of claim 1 wherein thedetector detects ions.
 7. The system of claim 1 wherein the detectordetects radioactivity or light.
 8. The system of claim 1 furthercomprising men for accelerating the desorbed analyte to the detector. 9.The system of claim 1 wherein the affinity reagent is immobilized bybinding to the probe surface.
 10. The system of claim 1 wherein theaffinity reagent is immobilized by binding to a solid phase placed onthe probe surface.
 11. The system of claim 1 formed of a material thatcomprises the affinity reagent.
 12. The system of claim 1 wherein theprobe surface is derivatized for covalent or non-covalent binding to theaffinity reagent.
 13. The system of claim 1 wherein the affinity reagentis bound to the analyte.
 14. The system of claim 1 wherein the affinityreagent is free of the analyte.
 15. The system of claim 1 wherein tilesurface comprises a plurality of different affinity reagents.
 16. Thesystem of claim 1 wherein the affinity reagent comprises a metal ion.17. The system of claim 1 wherein the affinity reagent comprises anucleic acid.
 18. The system of claim 1 wherein the affinity reagentcomprises a carbohydrate.
 19. The system of claim 1 wherein the affinityreagent comprises a polypeptide.
 20. The system of claim 1 wherein theaffinity reagent comprises an inhibitor of analyte function or amolecule that modifies analyte structure or function.
 21. The system ofclaim 1 wherein the surface comprises metal, metal coated with asynthetic polymer, glass, ceramic, a synthetic polymer or a mixturethereof.
 22. The system of claim 1 wherein the surface is adhered to theprobe magnetically.
 23. The system of claim 1 wherein the surface iscoated with a synthetic polymer.
 24. The system of claim 1 wherein theprobe comprises glass.
 25. The system of claim 1 wherein the probecomprises ceramic.
 26. The system of claim 1 wherein the probe comprisesa synthetic polymer.
 27. The system of claim 1 wherein the affinityreagents are comprised in spots arranged in a predetermined array. 28.The system of claim 12 wherein the affinity reagent is non-covalentlybound to the probe surface.
 29. The system of claim 12 wherein theaffinity reagent is covalently bound to the probe surface.
 30. Thesystem of claim 16 wherein the affinity reagent is a metal ion selectedfrom Cu(II) and Fe(III).
 31. The system of claim 16 wherein the metalion is bound to the probe surface though coordinate covalent bonds. 32.The system of claim 17 wherein the nucleic acid is DNA.
 33. The systemof claim 19 wherein the polypeptide is an antibody.
 34. The system ofclaim 19 wherein the polypeptide is a lectin.
 35. The system of claim 19wherein the polypeptide is an enzyme.
 36. The system of claim 19 whereinthe polypeptide is an inhibitor of analyte function.
 37. The method ofclaim 27 wherein the array comprises an array of spots from 0.005 to0.080 inches in diameter.
 38. A method for detecting an analytecomprising the steps of:a) providing a system comprising:(1) a removablyinsertable probe having a surface for presenting the analyte to anenergy source that emits energy capable of desorbing the analyte fromthe probe, and an immobilized affinity reagent on the probe surfacecapable of binding the analyte; (2) an energy source that directs energyto the probe surface for desorbing the analyte; and (3) a detector incommunication with the probe surface that detects the desorbed analyte;b) desorbing at least a portion of the analyte from the probe surface byexposing the analyte to energy from the energy source; and c) detectingthe desorbed analyte with the detector.
 39. The method of claim 38wherein the system is a laser desorption mass spectrometer wherein theenergy source emits laser light that ionizes the analyte to produce anion, the detector detects the ion and the stem further comprises meansfor accelerating the ion to the detector, and the method furthercomprises determining the mass of the ion.
 40. The method of claim 38further comprising before step (b) the step of modifying the analytechemically or enzymatically while deposited on the probe surface. 41.The method of claim 38 further comprising after step (a) the steps of:d)modeling the analyte chemically or enzymatically while deposited on theprobe surface; and e) repeating steps b) and c).
 42. The method of claim38 wherein the probe surface comprises an array of locations, eachlocation having at least one analyte deposited thereon; and step (b)comprises desorbing a first analyte from a first location in thearray;and wherein the method further comprises the stop of:d) desorbinga second analyte from a second location in the array; and e) detectingthe desorbed second analyte with the detector.
 43. The method of claim38 wherein the affinity reagent is immobilized by binding to the probesurface.
 44. The method of claim 38 wherein the affinity reagent isimmobilized by binding to a solid phase and the method comprises thesteps of:exposing the affinity reagent bound to the solid phase to theanalyte whereby the analyte binds to the affinity reagent; transferringthe solid phase to the probe surface; and removably inserting the probeinto the system.
 45. The method of claim 38 wherein the affinity reagentcomprises a nucleic acid.
 46. The method of claim 38 wherein the analytecomprises a peptide.
 47. The method of claim 38 wherein the analytecomprises a nucleic acid.
 48. The method of claim 38 wherein the analytecomprises a carbohydrate.
 49. The method of claim 39 further comprisingthe step of displaying the determined mass of the analyte.
 50. Themethod of claim 44 wherein the solid phase affinity reagent comprisesthe affinity reagent attached to a material selected from the groupconsisting of an electrically insulating material, a flexible material,an optically transparent material, a cross-linked polymer and abiopolymer.
 51. The method of claim 50 wherein the material is a bead.52. The method of claim 50 wherein the material is a cross-linkedpolymer or a biopolymer.
 53. The method of claim 50 wherein the materialis selected from the group consisting of agarose, dextran and cellulose.54. The method of claim 50 wherein the material is glass or a syntheticpolymer selected from polystyrene, polypropylene, polyethylene andpolycarbonate.